Cloning of honey bee allergen

ABSTRACT

The present invention relates to a recombinant polypeptide capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera having a homology of more than 70% to the amino acid sequence of SEQ ID NO: 2, which is the honey bee allergen Api m3 (acid phosphatase). The invention further relates to nucleic acid encoding the polypeptide, expression vectors, host cells and methods of preparing the polypeptide, as well as diagnostic and pharmaceutical uses thereof.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent application Ser. No. 11/301,329 filed Dec. 13, 2005, which claims the benefit under 35 USC 119(e) of U.S. provisional application 60/635,479, filed Dec. 14, 2004.

FIELD OF THE INVENTION

The present invention relates to a recombinant polypeptide capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera having a homology of more than 70% to the amino acid sequence of SEQ ID NO: 2, which is the honey bee allergen Api m3 (acid phosphatase). The invention further relates to nucleic acid encoding the polypeptide, expression vectors, host cells and methods of preparing the polypeptide, as well as diagnostic and pharmaceutical uses thereof.

SUMMARY OF THE INVENTION

It has long been recognised that allergies against insect venoms are relatively common. 4-5% of the German population react allergic to insect venoms. In Europe the relevant stinging insects are honey bees (Apis mellifera), wasps (Vespula spp.), bumble bees (Bombus spp.), hornets (Vespa crabo), midges, and horse flies (Helbing et al 2004, Eich-Wanger and Müller 1998). Bees, bumble bees, wasps, and hornets belong to the order Hymenoptera.

These social insects do not normally attack people, but will sting them in self defense if disturbed. Once stung, if the stinger remains in the skin, a honey bee is responsible, while, if no stinger is present, a wasp is likely to be the culprit. The female worker honey bee carries the stinger and dies soon after discharging a sting.

If a bee stings a vertebrate, the stinger will be detached from the insect, but the venom sack will still be attached to the stinger and if not removed, the whole venom volume (up to 50 μl) will be injected into the victim. Wasps can retract the stinger, and only inject about 20 μl venom.

The differences in stinging behavior are based on natural evolution. Bees collect nectar, whereas wasps and hornets are insect hunters. Therefore, bees need to protect the hive, even against vertebrates like mice or larger animals. The insect dies upon the sting, but will inject the maximum volume of venom, if the stinger is not removed. Wasps and hornets do not have such natural enemies.

Since it is easy to obtain sufficient quantities of material, honey bee venom has been well studied. Honey bee venom contains at least 18 active substances. Melittin, the most prevalent substance, is one of the most potent anti-inflammatory agents known (100 times more potent than hydrocortisone). Adolapin is another strong anti-inflammatory substance, and inhibits cyclooxygenase; it thus has analgesic activity as well. Apamin inhibits complement C3 activity, and blocks calcium-dependent potassium channels, thus enhancing nerve transmission. Other substances, such as Compound X, Hyaluronidase, Phospholipase A2, Histamine, and Mast Cell Degranulating Protein (MSDP), are involved in the inflammatory response to venom, with the softening of tissue and the facilitation of flow of the other substances. Finally, there are measurable amounts of the neurotransmitters Dopamine, Norepinephrine and Serotonin. The water content varies between 55-70%. The pH range is between 4.5-5.5. A summary of the components of bee venom is given in Table 1 (Dotimas and Hider 1987).

The LD50 dose, i.e., the amount of bee venom which causes 50% of the tested individuals to die, is 6 mg venom/kg body weight for mice and rats. This equals 40 stings/kg body weight. For hornets, this factor is around 154-180 stings/kg body weight. Bee venom is 1.7-1.5 more effective than those of hornets (Habermann 1974, Kulike 1986).

Honey bees and wasps of the Hymenoptera order are by far the most frequent cause of serious allergic reactions. Normally, at least more than 50 stings of a bee per children or 100 per adult are necessary to induce life threatening conditions (see above). In case of allergic persons, one sting can be enough to cause death by adverse immunological reactions.

This type of allergy is mediated by IgE antibodies which react to venom components. The possibility, therefore, exists that desensitization therapy by repeated and progressively increased doses of bee venom components would be successful. Several polypeptides from bee venom have been cloned and expressed as recombinant molecules (Sobotka et al 1974, Sobotka et al 1976, Hoffman and Shipman 1976, Kuchler et al 1989, Gmachl and Kreil 1993, Vlasak et al 1983, Hoffman et al 1977, Kettner et al 1999, King and Spangfort 2000). One component of bee venom, acid phosphatase, is one of the more potent allergic proteins (Arbesman et al 1976). Until now, no information about the complete gene sequence has been published and only initial studies on protein level have been made (Soldatova et al 2000, Barboni et al 1987, Hoffman 1996, Jacobsen and Hoffman 1995).

Barboni et al. (1987) describe two different proteins with acid phosphatase activity from honey bee venom, having a molecular weight of 45 and 96 kDa. Enzymatic activity is partly lost during purification in the gel filtration step. Other publications (Soldatova et al 2000, Barboni et al 1987, Jacobsen and Hoffman 1995) report contrasting data, teaching different fragments of the protein and the corresponding nucleic acid, and coming to different conclusions about the family of phosphatases that honey bee venom acid phosphatase might belong to, either prostatic phosphatases or lysosomal phosphatases. Soldatova et al. (2000) describe the incomplete cloning of a partial cDNA possibly encoding an acid phosphatase from honey bee venom. They report difficulties in cloning and obtaining the full length sequence and do not teach the sequence they seem to have cloned.

In light of the prior art, the person skilled in the art is therefore faced with the problem of providing a nucleic acid suitable for recombinant production of acid phosphatase (api m3) from the venom of an insect from the order Hymenoptera, in particular the honey bee, which can be used in such desensitization therapy as well as in diagnostic tests for the detection of allergy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of the isolated cDNA for Api m 3 in FASTA format (SEQ ID NO: 1).

FIG. 2 shows the predicted restriction enzyme pattern of the isolated cDNA for Api m 3.

FIG. 3 shows the predicted translated amino acid sequence of the isolated cDNA for Api m 3 (SEQ ID NO:2). The underlined peptides can be aligned to prior published fragments. See FIGS. 5A, 5B, and 6.

FIG. 4A shows a vector map of a preferred insect cell expression vector, pIB/Mel opt-H10 Api m 3. The vector was modified to include a N-terminal 10×histidine-tag, cleavable with factor Xa protease as well as the signal sequence of bee melittin for secreted expression. The gene of interest was cloned between the EcoR V and Sac II site. The gene comprises a stop codon at the 3′-end. The expressed protein should be secreted and will have a factor Xa cleavable 10× histidine-tag at the N-terminus.

FIG. 4B shows optimisation of the Kozak sequence for insect cell expression. The former sequence a) was changed into b) to be in accordance with the preferred translation initial sequence (G/A)NNATGG adding an alanine to the N-terminal sequence.

FIG. 4C shows a vector map of a preferred bacterial expression vector, pET26b(+) api 3 pro. The vector was modified to contain the gene of interest between the Sac I and Nde I site. The protein sequence was taken from the verified mammalian expression vector pIB/Mel opt-H10 api m 3.

FIG. 5A shows the sequence information of potential peptide fragments of acid phosphatase publicly known prior to this invention. Peptide fragments are listed in order of alignment to human and rat prostate phosphatase as published by Hoffman (1996). The alignment order of fragments to derived sequence is given in the second column. Positions of aligned peptide segments can be taken from FIG. 3 and FIG. 5B, as well as FIG. 6. Highlighted sequence segments in the third column show amino acids present in the Api m 3 sequence. The forth column shows the length of the published peptide fragments.

FIG. 5B shows the corrected alignment of peptides originally postulated by Hoffmann (1996) to Api m 3.

FIG. 6 shows a schematic alignment of peptides postulated by Hoffman et al. (1996) (A), in comparison to the corrected order after cloning and sequencing of the Api m 3 gene (B). It is obvious that the alignment differs from the published alignment with human and rat prostate phosphatase (Hoffman 1996). The published peptide fragments can not be aligned to match the sequence as would be expected. Firstly, the order of alignment positions is different from the publication. Secondly, some fragments, like fragments 1 and 7 partially align at different sites in the sequence, and therefore are not continuous peptides derived from a cDNA sequence. Furthermore, some published fragment sequences, like fragment 5, cannot be aligned at all. The scheme also shows the leader peptide and is not exact regarding the number of amino acids.

FIG. 7 depicts recombinant Api m 3 expression and purification. Shown is a 10% silver stained SDS-PAGE gel. Lane 1, protein molecular weight standards; lane 2, diluted bee venom; lane 3, purified recombinant Api m 3 derived from insect cell expression; lane 4, supernatant from cells stably transfected with recombinant Api m 3.

FIG. 8 Alignment of Api m 3 to acid phosphatase sequences. Shown is the alignment of cloned Api m 3 to different insect acid phosphatases with significant homology. The highest homology with 35% is found for Acph-1 from D. melanogaster. Amino acids necessary for acid phosphatase activity and for glycosylation are shaded in grey.

FIG. 9 Results from MALDI-TOF spectrometry in comparison with predicted tryptic fragments. Experimental data are in accordance with the prediction.

FIG. 10 shows the enzymatic activity of purified recombinant Api m 3. Shown is the acid phosphatase enzymatic activity of recombinant Api m 3 dependent on the amount of protein used. The experiment was performed according to Barboni et al (1987).

FIG. 11 shows an IgE immunoblot of pooled honey bee venom-reactive patient serum with recombinant Api m 3. Lane 1, protein molecular weight standards; lane 2, diluted bee venom; lane 3, purified recombinant Api m 3 derived from insect cell expression.

FIG. 12A Immunoreactivity of 59 individual patient sera with recombinant Api m 3. Shown are the results of an ELISA assay measuring the IgE antibody reactivity with Api m 3. FIG. 12A shows the results for an ELISA assay measuring the IgE antibody reactivity with Api m3 for 40 honey bee venom-sensitized patients (1-40; sIgE to honey bee venom ≧0.35 kU/L).

FIG. 12B shows the results for an ELISA assay measuring the IgE antibody reactivity with Api m 3 for 19 honey bee venom-negative patients (41-50; sIgE to honey bee venom <0.35 kU/L and to vespid venom ≧50 kU/L) (51-59; sIgE to honey bee and vespid venom <0.35 kU/L).

FIG. 12C shows an 8-point calibration ELISA standard for total human IgE (31.25; 62.5; 125; 250; 500; 1,000; 2,000; 4,000 pg/ml) for an ELISA assay measuring the IgE antibody reactivity with Api m 3.

FIG. 13 shows the detection of native Api m 3 with IgE from sera of honeybee venom-allergic individuals. Experimental conditions are described in Example 6. Shown are data after subtraction of background values.

FIG. 14 shows the detection of prokaryotically produced Api m 3 with IgE from sera of honeybee venom-allergic individuals. Experimental conditions are described in Example 6. Shown are data after subtraction of background values.

FIG. 15 shows the detection of Api m 3 produced in insect cells (HighFive insect cells in A and Sf9 insect cells in B) with IgE from sera of honeybee venom-allergic individuals. Experimental conditions are described in Example 6. Shown are data after subtraction of background values.

FIG. 16 shows the detection of Api m 3 produced in Sf9 and HighFive insect cells with IgE from sera of patients allergic to both honeybee and wasp venom. Experimental conditions are described in Example 6. Shown are data after subtraction of background values.

DETAILED DESCRIPTION

This problem is solved by the subject matter of the claims.

Specific immunotherapy (desensitization) approaches are well known in the state of the art. In principle, repeated treatments of allergic individuals with suitable, normally progressively increased doses of allergen diverts the immune response to one dominated by T cells that favour the production of IgG and IgA antibodies over production of IgE antibodies. The IgG and IgA antibodies are thought to desensitize the subject by binding to the small amounts of allergen normally encountered, and preventing the allergen from binding to IgE. Desensitization to honeybee venom is relatively successful (e.g., Hunt et al 1978).

However, there are serious limitations to the use of currently available allergen preparations for specific immunotherapy. While multiple studies have demonstrated that successful SIT requires administration of high doses of allergens, effective dosages are limited by potential systemic reactions. As a result, specific immunotherapy usually requires a treatment period of 2 to 3 years, over which the allergen preparation is administered at slowly increasing dosages followed by several injections of the final maintenance dose. Since the compliance of patients and doctors is very low due to the tedious and potentially harmful procedure, there is a need in the field for modified allergens capable of providing protection without the danger of serious side-effects.

In order to avoid undesirable systemic reactions on specific immunotherapy with natural allergens, there has been continued interest in the development of modified allergens with reduced allergenic activities for immunotherapy. In one approach T cell epitopes are used to modulate allergen-specific immune responses. It has been observed in vivo in mice for the allergen Fel d 1 (cat hair), Der p 1 (acarian: Dematophagoides pterissimus) and Bet v 1 (birch pollen) that the nasal, oral or subcutaneous administration of peptides carrying T cell epitopes of these allergens inhibits the activation of the specific T lymphocytes (Briner et al 1993; Hoyne et al 1993; Bauer et al 1997). Based on these results allergen peptide fragments capable of stimulating T lymphocytes in allergic patients were evaluated in clinical studies. In the case of the major honeybee venom allergen Api m 1 fragments 50-69 and 83-97 have been described as being active during a study comprising a single patient (Dhillon et al. 1992). In a study comprising forty patients (Carballido et al 1993) Api m 1 fragments 45-62, 81-92 and 113-124 proved to be active. However, these three fragments proved to be T cell epitopes for only 25 to 45% of the patients, pointing to the existence of other epitopes. Nevertheless, the three peptides have been used successfully for desensitization of five allergic patients whose T lymphocytes proliferated in the presence of these peptides (Müller et al 1998). No serious systemic effect was observed and the patients became tolerant to honeybee stings. This demonstrates the benefit of using peptides for desensitization. Therefore, there is a need in the field to identify peptide fragments of Api m 3 capable of stimulating T lymphocytes in patients allergic to honeybee venom.

In another approach, B cell epitopes of allergens are modified to decrease the risk of potential systemic reactions. B cell epitopes of poteinaceous allergens can include native protein structures (conformational or discontinuous or topographic epitopes), linear peptides (linear epitopes) and carbohydrates. The conformational type consists of amino acid residues which are spatially adjacent but may or may not be sequentially adjacent. The vast majority of IgE epitopes has been reported to be of the conformational type (King 1990). The linear type consists of only sequentially adjacent residues. However, even linear B cell epitopes are often conformation-dependent, and antibody-antigen interactions are improved when the epitope is displayed in the context of the folded protein. It is believed that the entire accessible surface of a protein molecule can be recognized as epitopes by the antigen receptor of B cells, although all epitopes are not necessarily recognized with equal likelihood (Benjamin et al 1984).

The aim of modification of B cell epitopes is to decrease the allergenicity while retaining its immunogenicity. Since allergenicity depends on the interaction of a multivalent allergen with basophil- or mast cell-bound IgE antibodies, allergenicity can be reduced by decreasing the density of B cell epitopes. One approach is by partial or complete denaturation of allergens on chemical modification because the vast majority of B cell epitopes are of the discontinuous type, being dependent on the native conformation of proteins. However, there are serious limitations to the use of such molecules. While linear T cell epitopes are preserved, the surface structure is not maintained and, thereby, the capacity of such molecules to stimulate an allergen-specific non IgE antibody response is severely limited. Similar considerations apply to an approach in which the accessibility of B cell epitopes is reduced by polymerization on formaldehyde or glutaraldehyde treatment or by attachment of non-immunogenic polymers. Usually near-complete loss of the discontinuous B cell epitopes occurs when allergens are modified with >100-fold reduction in allergenicity.

A more promising approach is to modify by site-directed mutagenesis identified discontinuous B cell epitopes recognized by IgE antibodies. While several IgE epitopes could be determined by mapping with synthetic overlapping peptides synthesized according to the allergen amino acid sequence, many relevant IgE epitopes could not be identified because peptides frequently fail to display conformations mimicking discontinuous epitopes. Programs have been developed for the prediction of both linear and conformational B cell epitopes (Zhang et al 2008). For example, DiscoTope is a method for discontinuous epitope prediction that uses protein 3D structural data as input. It is based on amino acid statistics, spatial information and surface accessibility for a set of discontinuous epitopes determined by X-ray crystallography of antibody-proteinaceous antigen-complexes. However, available data are limited and not suited for a reliable identification of epitopes of the conformational type on the Api m 3 molecule. There is no doubt that naturally occurring IgE antibodies represent ideal tools for structural analyses of IgE epitopes. However, the number of monoclonal allergen-specific IgE antibodies isolated from blood lymphocytes of allergic patients so far is extremely limited. In an alternative approach, animal derived monoclonal allergen-specific antibodies can be useful to identify IgE epitopes. For example, from a panel of mouse monoclonal antibodies that effectively inhibited binding of birch pollen allergen Bet v 1 to specific IgE, several monoclonal antibodies identified a continuous epitope within an exposed surface area of Bet v 1 that could be part of a discontinuous IgE epitope (Lebecque et al 1997) Provided such antibodies bind to Bet v 1 with high affinity, they represent useful tools for further structural analyses by X-ray diffraction of crystals obtained from allergen-antibody complexes. Although the surface area recognized by animal-derived allergen-specific antibodies may not be identical with that recognized by human IgE antibodies, both areas are closely related as indicated by the inhibition experiments. Therefore, structural information obtained from the analysis of such allergen-antibody complexes provide a valuable basis for the modification of IgE epotopes by site-directed mutagenesis. One problem of this approach, however, is the need of a panel of high affinity antibodies with different epitope specificities for each allergen to allow for a detailed analysis of the total spectrum of potential IgE epitopes. Assuming that a B cell epitope takes up an area of approximately 900 A², the vast majority of allergens is likely to display more than one IgE epitope. Therefore, there is a need in the field to develop high affinity Api m 3-specific antibody panels that are capable of inhibiting IgE binding.

Another serious problem associated with the design of a hypoallergenic Api m 3 molecule for an improved immunotherapy is the lack of understanding of the immune response that guarantees a lasting protection after specific immunotherapy. The aim to decrease the allergenicity of a given allergen while retaining its immunogenicity is widely accepted, but the term immunogenicity remains to be defined. Evaluation of modified recombinant allergens with a strongly reduced IgE reactivity that display the full spectrum of linear T cell epitopes but a different surface structure as compared to the corresponding natural allergen, have demonstrated that such molecules are capable of reducing specific IgE development towards the native allergen (Niederberger et al 2004; Karamloo et al 2005) However, a long lasting protective effect after treatment with these molecules has not been demonstrated. Apparently, the capacity of recombinant allergens to stimulate a long lasting protective allergen-specific non IgE antibody response requires also a surface structure that is closely related to that of the corresponding natural allergen. Since disruption of IgE epitopes is associated with a significant alteration of the surface structure, there is a need in the field to identify those surface structures of allergens that mediate an appropriate non-IgE response for a long lasting protection. There is a particular need in the field to identify those surface structures of Api m 3 that mediate an appropriate non-IgE response for a long lasting protection.

In particular, the present invention provides a nucleic acid encoding a polypeptide capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera wherein the polypeptide has a homology of more than 70% to the amino acid sequence of SEQ ID NO: 2 (note: “SEQ ID NO” relates to code <400> in the attached sequence listing under WIPO standard ST.25).

In one embodiment, the nucleic acid comprises a sequence homologous to the sequence of SEQ ID NO: 1 (derived from Apis mellifera), which is a naturally occurring homologous sequence from an other insect from the order Hymenoptera. The invention also refers to the recombinant proteins encoded by the nucleic acids of the invention.

Preferentially, the degree of homology to the amino acid sequence of SEQ ID NO: 2 is more than 75%, more than 80%, more than 85%, more than 90%, more than 95% or more than 99%. The sequence homology is determined using the clustal computer program available from the European Bioinformatics Institute (EBI). Most preferentially, the polypeptide encoded by the nucleic acid has the amino acid sequence of SEQ ID NO: 2. This polypeptide is designated Api m 3. In particular, the nucleic acid comprises or has the nucleotide sequence of SEQ ID NO: 1.

In the context of this application, sequence identity is used interchangeably with homology.

In the context of the present invention, the terms “polypeptide” and “protein” are used interchangeably, without any limitation as to the number of amino acids linked. The polypeptides may also comprise non-naturally occurring amino acids.

Throughout this specification, the polypeptides encoded by the nucleic acid of the invention have to be capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera. Of course, the skilled person understands that this binding takes place in the area of sequence identity or homology.

Following unsuccessful attempts to clone the full length sequence of Api m 3 following the common deduced primer strategy (cf. Soldatova et al., 2000) based on the peptide fragments found by Hoffmann, 1996, and their postulated sequence (Hoffmann, 1996). A completely different approach was chosen in the present invention. Using nucleic acid sequences derived from the peptide sequences published by Hoffman, small virtual probes of partial deduced sequences were constructed, which were used to scan the published bee genome for targets. Two regions on different truncated chromosome 16 sequences matched the probes. Scanning of these regions with bioinformatic tools revealed possible open reading frames and gene sequences. Scanning for a potential sequence cleavage site led to the putative N-terminus of the Api m 3 gene. Primers were then designed according to the proposed protein N- and C-terminus. These primers were used to amplify the gene from bee venom gland cDNA synthesized from total RNA with oligodT(20) primers. The amplification was successful and resulted in a DNA fragment of the expected size. The identity of the DNA was verified by sequencing, molecular weight calculation and alignment to the homologous acid phosphatases of human as well as rat sequences and the proposed peptide fragments. The protein identity was also verified.

From the resulting full length cDNA sequence, it is clear the classical approach had to fail, as Hoffman erred in several points. For example, he chose an incorrect alignment of the peptide fragments (cf. FIG. 5), erroneously thought that several peptides that are in fact separated by other peptides were contiguous, and postulated existence of a fragment that does not belong to the acid phosphatase. In fact, using primers based on peptides 1 and 7, as proposed by Hoffman, one would expect to amplify a short fragment covering only the peptide sequence around amino acids 200 and 230 of the consensus sequence.

The social insects from the order Hymenoptera that commonly interact with man are members of the superfamilies Apoidea and Vespoidea, bees and wasps (Hoffman 1996). The Vespoidea include the social wasps and hornets, Vespidae, as well as ants, Formicidae. Important wasps comprise yellowjackets of the genus Vespula, hornets of the genera Dolichovespula and Vespa and paper wasps of the genus Polistes. Bees comprise, e.g., honey bees, Apis mellifera, and bumble bees of the species Bombus terrestris. In the context of the present invention, an insect from the order Hymenoptera can be from any of these species, but according to a particular embodiment, the insect is a bee from the genus Apis. Most preferably, the bee is the honey bee, Apis mellifera.

Other species from the order Hymenoptera produce similar allergens with antigenic cross reactivity and a high degree of amino acid homology (Wypych et al 1989, Castro et al 1994, Hoffman et al 1988). Thus the present invention not only extends to the Api m3 allergen from Apis mellifera but also to homologous Hymenoptera allergens.

In particular, the polypeptides encoded by the nucleic acids of the invention have to be capable of binding to IgE from subjects allergic to venom of Apis mellifera. The subjects are commonly reactive to the Api m3 antigen, acid phosphatase from bee venom. For the purpose of testing, serum or purified IgE from such allergic subjects are contacted with the polypeptide, and specific binding of the polypeptide to the antibodies is detected. Such a test can, e.g., be an ELISA. For verifying the reactivity of the polypeptides with IgE antibodies, serum or IgE from several subjects are pooled, preferentially, from 5 to 20 subjects.

The nucleic acids of the invention can be either DNA or RNA.

In one embodiment, the invention also provides a nucleic acid, which is a fragment having a length of more than 255 nucleotides of a nucleic acid encoding a polypeptide having a homology of more than 70% to the amino acid sequence of SEQ ID NO: 2, wherein the fragment encodes a polypeptide capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera. Preferably, the nucleic acid is a fragment having a length of more than 255, more preferably of more than 600, more than 700 or more than 800 nucleotides of a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.

In another embodiment, a nucleic acid fragment (polynucleotide) is provided that comprises at least 15 contiguous nucleotides of the nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2, wherein the polynucleotide is selected from the group consisting of nucleotides 78 to 299, 348 to 437, 459 to 476, 555 to 671, 696 to 830 or 1086 to 1121 of said nucleic acid, wherein the numbering corresponds to the region encoding said polypeptide. Specifically, said nucleic acid has the nucleotide sequence shown in SEQ ID NO: 1. Preferentially, the nucleotides are from the region of nucleotides 555 to 671 or 696 to 830. Alternatively, the nucleic acids encode polypeptides that are capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera, and comprise at least 15, preferably at least 18, 21, 24, 27, 30, 45, 60 or more nucleotides of a nucleic acid more than 70%, more than 80% or more than 90% homologous or identical to the nucleic acid shown in SEQ ID NO: 1, except for the nucleic acids from the group consisting of nucleotides 1 to 104, 189 to 142, 300 to 347, 426 to 449, 504 to 530, 672 to 719 and 774 to 1031 of the nucleic acid shown in SEQ ID NO: 1 or except for the nucleic acids encoding the polypeptides shown in FIG. 5. Additionally, such nucleic acids consisting of nucleotides 1 to 77, 438 to 458, 477 to 494, 504 to 554, 672 to 695, and 831 to 1085 of the nucleic acid shown in SEQ ID NO: 1 are provided.

Preferentially, the nucleic acid comprises 15 to 240, 15 to 90, 18 to 60, 21 to 30, more preferably at least 18, 21, 24, 27, 30, 60, 90 or more contiguous nucleotides from the above regions.

Alternatively, a nucleic acid is provided which encodes a polypeptide having more than 70% homology to the polypeptide encoded by said at least 15 contiguous nucleotides, wherein the polypeptide is capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera. In particular, this polypeptide comprises at least 5, preferably at least 6, 7, 8, 9, 10, 15, 20 or more amino acids of a polypeptide more than 70%, more than 80% or more than 90% homologous or identical to a polypeptide selected from the group consisting of amino acid 26 to 99, 116 to 145, 153 to 158, 185 to 223, 232 to 276 or 362 to 373 of the polypeptide shown in SEQ ID NO: 2. Alternatively, the polypeptides encoded by the nucleic acids are capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera, and comprise at least 5, preferably at least 6, 7, 8, 9, 10, 15, 20 or more amino acids of a polypeptide more than 70%, more than 80% or more than 90% homologous or identical to the polypeptide shown in SEQ ID NO: 2, except for the polypeptides from the group consisting of amino acids 1 to 34, 63 to 80, 100 to 115, 142 to 149, 168 to 176, 224-239 and 258 to 343 of the polypeptide shown in SEQ ID NO: 2 or except for the polypeptides shown in FIG. 5. Additionally, such polypeptides consisting of amino acids 1 to 25, 146 to 152, 159 to 164, 168 to 184, 224 to 231, and 277 to 361 are encoded by the nucleic acids.

In one embodiment, the invention also provides a polypeptide encoded by a nucleic acid of the invention. Preferentially, the polypeptide is a full length acid phosphatase from the venom of an insect from the order Hymenoptera. In particular, the polypeptide has an homology of more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95% or more than 99% to the amino acid sequence of SEQ ID NO: 2. Most preferred is a polypeptide having the amino acid sequence of SEQ ID NO: 2. Although not essential, it is preferred that the polypeptide has acid phosphatase activity. This activity can be tested, e.g., according to the method described by Barboni et al 1987.

Alternatively, the polypeptide is a fragment of the full length protein capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera having a length of more than 85, more than 200 or more than 250 amino acids. Other fragments are provided, wherein the polypeptides are capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera, and comprise at least 5, preferably at least 6, 7, 8, 9, 10, 15, 20 or more amino acids of a polypeptide more than 70%, more than 80% or more than 90% homologous or identical to a polypeptide selected from the group consisting of amino acid 26 to 99, 116 to 145, 153 to 158, 185 to 223, 232 to 276 or 362 to 373 of the polypeptide shown in SEQ ID NO: 2. Alternatively, the polypeptides are capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera, and comprise at least 5, preferably at least 6, 7, 8, 9, 10, 15, 20 or more amino acids of a polypeptide more than 70%, more than 80% or more than 90% homologous or identical to the polypeptide shown in SEQ ID NO: 2, except for the polypeptides from the group consisting of amino acids 1 to 34, 63 to 80, 100 to 115, 142 to 149, 168 to 176, 224-239 and 258 to 343 of the polypeptide shown in SEQ ID NO: 2 or except for the polypeptides shown in FIG. 5. Additionally, such polypeptides consisting of amino acids 1 to 25, 146 to 152, 159 to 164, 168 to 184, 224 to 231, and 277 to 361 are provided.

In a preferred embodiment, the invention provides T-cell epitope-containing oligopeptides of at least 9 amino acids corresponding to a consecutive amino acid sequence within the Api m 3 molecule wherein the peptides are capable of stimulating T-cells of subjects allergic to Api m 3. Such peptides of the invention are preferably immunomodulatory peptides as well in that they induce T-cell energy when administered to a subject allergic to Api m 3, or otherwise affect the immune response of the subject. Preferably, the amino acid sequence of the T-cell epitope-containing oligopeptide corresponds to a consecutive amino acid sequence of a polypeptide having the amino acid sequence of SEQ ID NO: 2, wherein the T-cell epitope-containing oligonucleotide is selected from the group consisting of 15 contiguous amino acid residues as defined in Tables 3 and 4 of said polypeptide, wherein the numbering corresponds to the region of said polypeptide.

T-cell stimulating activity can be tested by culturing T-cells obtained from an individual sensitive to the Api m 3 polypeptide, fragments, and analogs thereof described herein, with the Api m 3 polypeptide, fragments, and analogs thereof, and determining the presence or absence of proliferation by the T-cells in response to the peptide as measured by, for example, uptake of tritiated thymidine. Stimulation indices for responses by T-cells to peptides useful in methods of the invention can be calculated as the maximum counts per minute (cpm) taken up in response to the peptide divided by the cpm of the control medium. For example, a peptide derived from a protein allergen may have a stimulation index of about 2.0. As a result, a stimulation index of at least 2.0 is generally considered positive for purposes of defining peptides useful as immunotherapeutic agents. Preferred peptides have a stimulation index of at least 2.5, more preferably at least 3.5 and most preferably at least 5.0.

Preferably, the polypeptide of the invention is recombinantly expressed. This has the advantage, e.g., that the polypeptide can be expressed as a fusion protein linked to an additional polypeptide. For example, the polypeptide or fusion protein is attached to a signal sequence ensuring its secretion into the extracellular space or supernatant of the cultured cells, where appropriate. Due to novel techniques in molecular biology, the use of recombinant proteins in therapy and diagnostics is expected to increase the efficiency and diagnostic value in these medical applications (King 1990, Müller 2001, Müller 2002).

Depending on the host cell producing the recombinant protein, the protein is glycosylated (after expression in mammalian or yeast cells) or non-glycosylated (after expression in bacterial cells). The glycosylation pattern can vary depending on the host cell used, and can thus differ from the glycosylation pattern of natural acid phosphatase isolated from bee venom. In one alternative, the glycosylation pattern is identical to the glycosylation pattern of acid phosphatase isolated from bee venom. Glycosylation can have profound effects on the binding of specific antibodies.

When expressed in bacterial cells, the polypeptide of the invention lacks glycosylation. The protein thus differs from the native protein in respect to epitope presentation, and potentiality for folding and functionality. It was shown that carbohydrates can represent IgE epitopes and contribute to observed non-specific cross-reactivity of allergens, e.g., between bee and wasp proteins, due to similar features of the carbohydrate chains (Huby et al 2000, Tretter et al 1993, Hemmer et al 2004). The cross-reactivity is one reason for false positive results in in vitro immunological tests (Petersen and Mundt 2001). Expression of the non-glycosylated polypeptide eliminates these false positives, and can therefore be used to advantage in diagnostic and therapeutic applications.

The glycosylation pattern in eukaryotic cells other than insect cells, e.g., in mammalian cells, also varies from the glycosylation pattern of the native protein (Jenkins et al 1996). Even in insect cells, the glycosylation pattern is likely to be different due to overexpression of the protein.

Sequence analysis of Api m 3 shows that the protein comprises three putative glycosylation sites of the sequence Asn-Xaa-Ser/Thr. In one embodiment, the polypeptides of the invention comprise mutated glycosylation sites instead of glycosylation sites. In particular, in a mutated glycosylation site, the Asparagine (Asn) in the glycosylation site(s) can be exchanged against any other amino acid, preferably against Glutamine (Gln) (Elbein et al 1991). Alternatively, in a mutated glycosylation site, the Serine (Ser) can be exchanged against another amino acid or deleted. Accordingly, the invention also provides a nucleic acid encoding a polypeptide of the invention comprising at least one, preferably 2, or 3 mutated glycosylation sites instead of glycosylation sites. Most preferably, all glycosylation sites are mutated.

Using native Api m 3 in diagnostic assays for detecting allergy, e.g., to bee or wasp venom, cross-reactivity is a big problem. Based on the state of the art using native purified Api m 3 as an antigen in diagnostic tests, the skilled person was unable to differentiate between patients that had been sensitized to bee venom, patients that had been sensitized to wasp venom, and patients that had been sensitized to both. This differentiation is important, because, in case, e.g., a bee allergy is incorrectly diagnosed, a desensibilization therapy might be prescribed which then in fact serves to sensitize the patient to epitopes of bee allergen he was not previously allergic to.

The present invention now allows preparation of recombinant proteins that are useful in diagnostic tests to differentiate between such patients, because it provides recombinant antigens that are not bound by sera of some patients previously diagnosed as allergic to native Api m3 of honey bee venom. The expressed proteins exhibit epitopes that react with IgE antibodies to native Api m 3, but they do not react with all IgE antibodies that bind to native Api m 3.

As mentioned above, cross-reactivity is mainly due to the glycosylation of the bee protein, the sugar patterns being similar to glycosylation of e.g., wasp proteins.

The inventors have shown that unglycosylated Api m 3, e.g., expressed in procaryotes, provides IgE epitopes as the proteinaceous part of native Api m 3. Furthermore, as shown in FIG. 14, both prokaryotic Api m 3-fusion constructs exhibit a different reactivity to IgE in sera from patients with honeybee venom allergy. Based on these data both constructs provide a different set of IgE epitopes indicating a different folding structure. Such fusion proteins are extremely valuable in assessing sensitization of patients to the proteinaceous part of Api m 3. The differential reactivity of both fusion proteins to IgE antibodies as compared to the reactivity of native Api m 3 purified from bee venom, clearly demonstrates that recombinant, e.g., non-glycosylated Api m 3 fusion proteins provide novel means to eliminate carbohydrate mediated cross-reactivity, thereby eliminating potentially false positives in the diagnosis of honeybee venom allergy.

The results shown in the examples also demonstrate, that, similarly, recombinant Api m 3 molecules expressed in HighFive and SF9 insect cells are recognized to a different extent by IgE in sera from patients allergic to both honeybee and wasp venom.

As explained above, recombinant Api m 3 molecules expressed in insect cells (e.g., HighFive cells and SF9 cells) are glycosylated, but the glycosylation pattern provided by both insect cell lines to Api m 3, exhibits significant differences. As shown in FIG. 15, the two glycosylated Api m 3 molecules expressed in HighFive insect cells and SF9 insect cells exhibit a different reactivity to IgE in sera from patients with honeybee venom allergy. Furthermore, FIG. 16 demonstrates that both molecules are recognized to a different extent by IgE in sera from patients allergic to both honeybee and wasp venom. This observation is important, since both molecules allow an improved evaluation of carbohydrate based cross-reactivity of IgE antibodies.

In contrast to the data obtained with Api m 3 produced in HighFive insect cells, IgE antibodies in sera from patients allergic to both honeybee and wasp venom recognize Api m 3 produced in SF9 insect cells to a much lesser extent (see FIG. 16). Although Api m 3 produced in SF9 insect cells is also recognized by IgE in 15 of 23 (65%) of these sera, the IgE reactivity is very low as compared to the IgE reactivity towards Api m 3 produced in HighFive insect cells. The residual reactivity of IgE antibodies in sera from patients allergic to both honeybee and wasp venom could be due to those patients possessing IgE antibodies recognizing the proteinaceous part of Api m 3. The recombinant proteins produced according to the invention thus for the first time allow differentiation between allergic patients having antibodies binding to different epitopes of the antigen, which can lead to clearer diagnosis of allergies and potential cross-reactivity.

The present invention also relates to an expression vector comprising a nucleic acid of the invention operationally linked to an expression control sequence. In one alternative, the nucleic acid is linked in frame to a nucleic acid encoding an additional polypeptide, so the expression vector can be used for expression of a fusion protein. The additional polypeptide can be selected from the group comprising a poly-Histidine tag (His tag), glutathione-S-transferase, β-galactosidase, a cytokine, and an IgG-Fc. In particular, tags that simplify purification of the recombinant protein, e.g., a His tag, are employed. Such a tag may be cleaved off after purification of the protein.

Alternatively, it can be beneficial for therapeutic applications to express the polypeptide of the invention linked to a therapeutic polypeptide, e.g. a cytokine. For example, a fusion protein with a cytokine enhancing T_(H)1 and downregulating T_(H)2 responses or inducing class switch to IgG, such as IFN-γ, IL-10, IL-12 or TGF-β, can improve efficiency of desensitization. If the expression vector is used for gene therapy, it is envisaged to use sequences rich in CpG (unmethylated cytosine guanidine dinucleotides), which promote T_(H)1 responses. Additionally or alternatively, the polypeptide of the invention can be linked to another polypeptide or protein, such as in the form of a fusion protein or as separate proteins expressed by the same vector. Preferably, the further polypeptides or proteins are other Hymenoptera venom proteins or antigenic fragments thereof.

The expression vector can be suitable for expression in different cell types, such as bacterial, yeast or mammalian cells. Preferentially, the vector is suitable for expression in insect cells, e.g., HighFive insect cells (Invitrogen, Karlsruhe, Germany). Alternatively, especially for gene therapy applications, the vector is suitable for expression in human cells. In this context, the expression of the encoded polypeptide can be directed by the choice of a suitable expression control sequence, e.g., an expression control sequence mainly or specifically operational in different cell types, such as lymphoid cells, for example dendritic cells, B cells or macrophages.

In one embodiment of the invention, the expression vector is pIB/V5-His (Invitrogen, Karlsruhe, Germany, Invitrogen Manual: InsectSelect BSD System with pIB/V5-His, Version G, 30 May 2003).

In particular, the vector can be pIB/MeI opt-H10-Api m3, comprising the Api m3 cDNA sequence (SEQ ID NO: 1), which was modified to facilitate isolation and purification. A melittin signal sequence for secretion of the recombinant protein was added and the Kozak sequence was optimised for higher expression rates in insect cells (see FIG. 4 and Example 2). Alternatively, other signal sequences can be used for secretion of the protein. The expression vector can also be a different plasmid or a viral, e.g., baculoviral or adenoviral, vector. The expression vector further comprises a stop codon and a polyadenylation signal.

The present invention further relates to a host cell comprising said expression vector. This host cell can be a bacterial, yeast or mammalian cell, in particular an insect cell.

A method of producing a polypeptide encoded by a nucleic acid of the invention is provided, wherein the host cell is cultured under appropriate conditions for expression of said polypeptide and said polypeptide is purified. If the polypeptide is a fusion protein with a fusion partner facilitating purification, e.g., a His Tag or a GST-tag, a corresponding affinity column can be used for purification, e.g., a Ni²⁺ or glutathione affinity column. For purification of an IgG fusion protein, a protein A or protein G column is suitable.

The expression vector of the invention can be used for the preparation of a pharmaceutical composition for treating subjects allergic to the venom of an insect from the order Hymenoptera. Treatment regimens using gene therapy approaches to desensitization are known in the state of the art (e.g., Sudowe et al 2002).

The present invention also relates to a mutant Api m 3 molecule comprising a reduced IgE binding capacity with limited impairment of the residual surface structure important for IgG and IgA immunological responses.

In one embodiment, the present invention provides methods for identification and modification via site-directed mutagenesis of those amino acid residues involved in the interaction of the polypeptides of this invention with human IgE, IgG and IgA antibodies. In particular, the present invention provides compositions comprising recombinant antibodies wherein each composition is capable of binding to all epitopes recognized by human IgE, IgG (including IgG4) and IgA antibodies, a method of obtaining such a composition and the use of individual antibodies of such a composition as tools for the design of a hypoallergenic Api m 3 molecule for specific immunotherapy.

In a specific embodiment, antibody compositions capable of binding to all epitopes of the Api m 3 polypeptide, fragments and analogs thereof that are recognized by human IgE antibodies, are utilized to identify and modify by site-directed mutagenesis those amino acid residues involved in the interaction with allergen-specific human IgE antibodies, thereby eliminating or decreasing the allergenicity of the Api m 3 polypeptide, fragments and analogs thereof in a structure-based approach. By site-directed mutagenesis of amino acid residues essential for the allergen-IgE antibody interaction, IgE epitopes are eliminated with minimal impairment of the residual surface structure important for a non-IgE immunological response.

In another specific embodiment, antibody compositions capable of binding to all epitopes of the Api m 3 polypeptide that are recognized by human IgG antibodies, including IgG4 antibodies, and IgA antibodies are utilized to maintain those structures that mediate an appropriate non IgE response for a long lasting protection after specific immunotherapy (SIT). This rational is based on the recent observation that immune deviation towards T regulatory (Treg) cells is an essential step in successful SIT (for a review, see Jutel et al 2006). Treg cells are defined by their ability to produce high levels of IL-10 and TGF-β and to suppress naive and memory T helper type 1 and 2 responses. There is now clear evidence that IL-10- and/or TGF-β-producing type 1 T regulatory cells are generated in humans during the early course of SIT. Since Treg cells have been shown to differentiate from naive T cells in the periphery upon encountering antigens present at high concentrations, it can be assumed that Treg cells are also induced by high and increasing doses of allergens. Most important is the fact that IL-10 and TGF-β suppress directly or indirectly effector cells of allergic inflammation such as basophils and mast cells, induce the production of non-inflammatory immunoglobulin isotypes (IgG and IgA) and suppress IgE production. Based on these observations, antibody compositions capable of binding to all epitopes of the Api m 3 polypeptide, fragments and analogs thereof that are recognized by human IgG, particularly by human IgG4, and by IgA antibodies are utilized to identify and maintain those amino acid residues involved in the interaction with allergen-specific human IgG and IgA antibodies.

In the context of this invention, the term “epitope recognized by human IgE (IgE epitope), human IgG (IgG epitope), including human IgG4 (IgG4 epitope), or human IgA (IgA epitope)”, or relates to the surface area of an allergen that is in contact to these antibodies upon binding to the allergen. It also relates to the surface area of the allergen that is in contact with an antibody construct comprised in the composition of the invention, that overlaps with the first-mentioned IgE epitope, IgG epitope, including IgG4 epitope, or IgA epitope”, so binding of the antibody construct can inhibit binding of the human IgE, human IgG, including human IgG4; or human IgA from the sera of patients allergic to the allergen (IgE related epitopes, IgG-related epitopes, IgG4 related epitopes, IgA-related epitopes). Preferably, the epitopes overlap by 20% or more, 50% or more, 60% or more, 70% or more, or 80% or more. Most preferably, the epitopes overlap by 90 or 95% or more or are identical. With reference to the number of epitopes of an allergen, the first-mentioned epitopes and the related epitopes are considered to represent the same epitopes.

For an estimation of the number of antibodies sufficient for binding to all epitopes recognized by human IgE, human IgG (including human IgG4), or human IgA antibodies on the Api m 3 polypeptide, it is important to know the approximate number of possible B cell epitopes per allergen. Therefore, methods for estimating the number of B cell epitopes per allergen have been developed. These methods are based on the following parameters:

a) Calculation of the surface of structurally characterized allergens in A²: The solvent accessible surfaces of proteins can be calculated with the aid of POPS (parameter optimized surfaces) according to Fraternali and Cavallo (2002).

b) Surface area of B-cell epitopes in A²: At the moment, one co-crystallization of allergen and antibody is available only, namely for the allergen Bet v 1 and a murine allergen-specific Fab-fragment. The surface area of this discontinuous epitope is 931 A² (Mirza et al 2000). This correlates well with the area of other B cell epitopes (circa 2×3 nm).

In Table 5, the surface of allergens for which structural data is available in the protein data bank (PDB) was calculated with the aid of a molecule of water. Under the assumption that a B cell epitope takes up an area of 950 A², the maximal possible number of B cell epitopes for an allergen (without differentiation for IgE epitopes, IgG epitopes, or IgA epitopes) was determined. The number calculated in this way is much too high, but can be considered to provide an upper limit for the number of necessary antibody constructs for an allergen. On the basis of this data, an approximate relation between molecular weight and potential B cell epitopes was calculated. The mean value of the upper limit for potential B cell epitopes is approx. 0.5 B cell epitopes per 1 kDa.

Table 6 summarizes allergens that have been examined for IgE binding structures with overlapping oligopeptides. Utilizing overlapping oligopeptides (e.g., decapeptides), more potential IgE epitopes are identified than exist in reality, as the majority of IgE epitopes are discontinuous epitopes composed of at least two different areas of the molecule brought together by folding. Different relevant allergens, such as Phospholipase A2 and the birch pollen allergens Bet v1, Bet v3 and Bet v4 exclusively have discontinuous epitopes (Valenta et al 1998). Since the identified linear epitopes probably only form part of these discontinuous epitopes, for estimation of the number of epitopes it is supposed that at least three linear IgE binding epitopes are, as partial structures, involved in forming a discontinuous IgE epitope. Therefore, the number of identified IgE binding peptides has been divided by three and related to the molecular weight of the allergen. A number of 0.06 to 0.19 epitopes per 1 kDa calculated on the basis of linear IgE binding peptides is preferred. The best estimation is possible on the basis of the number of 0.12 IgE epitopes per 1 kDa, which is possibly still too high but could be considered realistic. The preferred compositions correlate well with known data for Bet v 2 (17.4 kDa), which can be bound by three different monoclonal Fab fragments (without differentiating between IgG epitopes, IgE epitopes, or IgA epitopes; Valenta et al 1998). Bet v 2 has at least two IgE epitopes, since it can induce in vivo cross-linking of surface-bound IgE antibodies.

The plurality of Api m 3-specific monoclonal antibodies can be generated from different sources. Naturally occurring IgE antibodies represent ideal tools for structural analyses of IgE epitopes, but their availability is limited. Cloning and selecting allergen-specific IgE antibodies from the immune repertoire of peripheral blood mononuclear cells of allergic donors is extremely difficult. The low number of IgE-secreting B cells in the peripheral blood of allergic patients (MacKenzie and Dosch 1989) seriously hampers this approach for generating monoclonal IgE antibodies. Cloning and selecting Api m 3-specific IgG antibodies, including IgG4 antibodies, or IgA antibodies from the immune repertoire of peripheral mononuclear cells of allergic donors may be less difficult due to the significantly higher number of IgG- or IgA-secreting B cells in the peripheral blood of allergic patients as compared to IgE-secreting B cells. Currently, however, the availability of human monoclonal allergen-specific IgG4 or IgA antibodies is limited.

Semisynthetic or synthetic immunolibraries (e.g., scFv or Fab format) provide a high degree of variability and, thereby, a valuable alternative for generating the required plurality of Ves v 4-specific monoclonal antibody fragments. However, newly generated immunolibraries derived from animals (mammalian species as well as avian species) after immunization with the Api m 3 polypeptide or fragments thereof provide a significantly higher number of Api m 3-specific variable antibody domains and, thereby, an increased probability for the selection of the required plurality of Api m 3-specific high affinity monoclonal antibody fragments. In a preferred embodiment a combination of immunolibraries derived from avian and mammalian species after immunization with the Api m 3 polypeptide or fragments thereof are used. The phylogenetic difference between avian and mammalian species provides access to a different antibody repertoire than the traditional mammalian antibodies. IgY antibodies recognize other epitopes than mammalian antibodies. Therefore, a combination of immunolibraries from avian and mammalian species provides a significant advantage for generating a plurality of Ves v 4-specific high affinity monoclonal antibodies. If it should—unexpectedly—be found that the combination of all antibodies capable of binding to the Api m 3 polypeptide is not sufficient to effect essentially complete inhibition of binding of Api m 3 to antibodies in a pool serum of patients allergic to said allergen or obtained from said sera, it is recommended to additionally use further antibodies from a different library. Methods for generating immunolibraries are known in the art (e.g., Steinberger et al 1996; Edwards et al 2002; Powers et al 2001; Boel et al 2000).

Each antibody composition is obtainable by a method for generating a composition comprising recombinant antibodies, comprising steps of

a) Generation a plurality of allergen-specific antibodies capable of binding to the Api m 3 polypeptide,

b) combining all generated antibodies and testing whether essentially complete inhibition of binding of the Api m 3 polypeptide to IgE, IgG (including IgG4), and IgA antibodies; in a pool serum of patients allergic to said allergen or obtained from said serum is achieved,

c) in case essentially complete inhibition is not achieved in step b), steps a) and b) are repeated;

d) in case essentially complete inhibition is achieved, the number of antibodies is reduced to the minimal number of antibodies sufficient for essentially complete inhibition by a method wherein

i) groups of the antibodies obtained in step a) are generated, comprising different numbers and combinations of antibodies;

ii) said groups are tested for essentially complete inhibition of binding of the Api m 3 polypeptide to IgE, IgG (including IgG4), and IgA antibodies, in a pool serum of patients allergic to said allergen or obtained from said sera;

iii) wherein, in case one or more group effects essentially complete inhibition in step ii), steps i) and ii) are repeated with sub-combinations of the antibodies from said group or groups until the minimal number of antibodies in said group or groups is identified which effects essentially complete inhibition;

iv) wherein, in case essentially complete inhibition is not achieved in step ii) or iii), steps i), ii) and iii) are repeated with different groups of antibodies;

and wherein the group identified in step d), iii) is said composition. It is preferred that the composition comprises the minimal number of antibodies necessary and sufficient for binding to all epitopes recognized by human IgE, human IgG (including human IgG4), and human IgA antibodies; on the Api m 3 polypeptide. Additional antibodies may, however, be added.

In the method of the invention, inhibition of binding of the Api m 3 polypeptide to IgE can be determined by incubating IgE antibodies in a pool serum of patients allergic to the Api m 3 polypeptide or antibodies obtained from said serum with human basophils after stripping of said basophils, and with or without preincubation of the Api m 3 polypeptide with the recombinant antibodies or recombinant antibody fragments or, for comparison, antibodies in a pool serum of patients allergic to said allergen or obtained from said serum, contacting said basophils with said allergen, and detecting release of histamine.

Alternatively, inhibition can be determined by contacting anti IgE antibodies immobilized on a carrier with antibodies in a pool serum of patients allergic to the Api m 3 polypeptide or obtained from said serum, and, with or without preincubation of labelled Api m 3 polypeptide with the recombinant antibodies or recombinant antibody fragments or, for comparison, antibodies in a pool serum of patients allergic to said allergen or obtained from said serum, contacting the carrier with said allergen and detecting binding of the labelled Api m 3 polypeptide to the carrier. For this purpose, the Api m 3 polypeptide can be labelled with an enzyme, a radioisotope, biotin or a fluorescent marker.

In the method of the invention, inhibition of binding of the Api m 3 polypeptide to IgG can be determined by contacting anti IgG antibodies immobilized on a carrier with antibodies in a pool serum of patients allergic to the Api m 3 polypeptide or obtained from said serum, and, with or without preincubation of labelled Api m 3 polypeptide with the recombinant antibodies or recombinant antibody fragments or, for comparison, antibodies in a pool serum of patients allergic to said allergen or obtained from said serum, contacting the carrier with said allergen and detecting binding of the labelled Api m 3 polypeptide to the carrier. For this purpose, the Api m 3 polypeptide can be labelled with an enzyme, a radioisotope, biotin or a fluorescent marker.

In the method of the invention, inhibition of binding of the Api m 3 polypeptide to IgG4 can be determined by contacting anti IgG4 antibodies immobilized on a carrier with antibodies in a pool serum of patients allergic to the Api m 3 polypeptide or obtained from said serum, and, with or without preincubation of labelled Api m 3 polypeptide with the recombinant antibodies or recombinant antibody fragments or, for comparison, antibodies in a pool serum of patients allergic to said allergen or obtained from said serum, contacting the carrier with said allergen and detecting binding of the labelled Api m 3 polypeptide to the carrier. For this purpose, the Api m 3 polypeptide can be labelled with an enzyme, a radioisotope, biotin or a fluorescent marker.

In the method of the invention, inhibition of binding of the Api m 3 polypeptide to IgA can be determined by contacting anti IgA antibodies immobilized on a carrier with antibodies in a pool serum of patients allergic to the Api m 3 polypeptide or obtained from said serum, and, with or without preincubation of labelled Api m 3 polypeptide with the recombinant antibodies or recombinant antibody fragments or, for comparison, antibodies in a pool serum of patients allergic to said allergen or obtained from said serum, contacting the carrier with said allergen and detecting binding of the labelled Api m 3 polypeptide to the carrier. For this purpose, the Api m 3 polypeptide can be labelled with an enzyme, a radioisotope, biotin or a fluorescent marker.

The inhibition by recombinant antibodies or recombinant antibody fragments is considered essentially complete if it is comparable to the inhibition by IgE, IgG (including IgG4), or IgA antibodies in a pool serum of patients allergic to the Api m 3 polypeptide or obtained from said serum, i.e. if it varies from that inhibition by 20% or less, preferably by 10% or less, or most preferably, by 5% or less.

The pool serum used in the present invention comprises serum from several patients allergic to the Api m 3 polypeptide. Preferably, said pool serum comprises the antibodies from the sera of at least 5 patients, at least 10 patients or at least 15 patients allergic to said allergen. For IgE inhibition experiments, patients are preferred that are highly sensitized to the allergen. For IgG, IgG4, and IgA inhibition experiments, patients after successful SIT are preferred.

IgE antibodies can be obtained from the pool serum, e.g., by affinity chromatography using anti-human IgE antibodies. Preferably, IgG antibodies are removed from the pool serum, e.g. by pre-treatment with a protein A matrix, such as a protein A column. This step, however, is not essential, as sera of allergic patients in all probability only contain relatively low amounts of allergen-specific IgG antibodies, even though the serum level of IgG is about 10.000 times higher than the serum level of IgE. For example, serum obtained from a birch pollen allergic patients which was purified by affinity chromatography on immobilized Bet v 1, did not contain significant quantities of allergen specific IgG antibodies (Ganglberger et al 2000). Human IgG4 and human IgA antibodies can also be obtained from the pool serum by affinity chromatography using anti-human IgG4 or anti-human IgA antibodies.

The individual antibodies of a generated composition are used for structural analyses of IgE, IgG (including IgG4), and IgA epitopes. Since each composition effects essentially complete inhibition of binding of the Api m 3-polypeptide to patient-derived IgE, IgG (including IgG4), and IgA antibodies, the individual antibodies of each generated composition are capable of identifying all epitopes on the Api m 3 polypeptide that are accessible for patient-derived IgE, IgG (including IgG4), and IgA antibodies.

According to the present invention the most potent Api m 3-related hypoallergenic molecule for specific immunotherapy is an allergen that does not exhibit allergenicity, contains an array of T cell epitopes that is comparable to that of the corresponding natural allergen, and displays a surface structure that is recognized by human IgG, particularly by human IgG4, and IgA antibodies with specificity for the corresponding natural allergen. For the design of such a molecule, the individual antibodies of the different antibody compositions are essential to maintain IgG epitopes and IgA epitopes upon modification of the IgE epitopes by a structure-based approach.

In specific embodiments, the present invention provides methods for decreasing the allergenicity (IgE reactivity) of the polypeptides of this invention in a structure-based approach via mutagenesis of IgE epitopes with limited impairment of the residual surface structure important for IgG and IgA immunological responses. In a preferred embodiment, the allergenicity of the polypeptides of this invention is reduced by at least 50% while at least 50% of IgG epitopes and IgA epitopes are maintained. In a more preferred embodiment, the allergenicity of the polypeptides of this invention is reduced by at least 70% while at least 50% of IgG epitopes and IgA epitopes are maintained. In a most preferred embodiment allergenicity is reduced by at least 90% while at least 50% of IgG epitopes are maintained.

In the context of this invention, allergenicity is defined as the capability of a proteinaceous allergen to bind human IgE antibodies. Antigenicity in the context of this invention is defined as the capability of a proteinaceous allergen to bind human IgG (including IgG4) and IgA antibodies.

The invention thus also provides a method of treating subjects allergic to the venom of an insect from the order Hymenoptera comprising administering to a subject with such an allergy a protein/polypeptide of the invention.

As used herein, “subject” encompasses human subjects (patients), grown-ups as well as children, and animals.

A pharmaceutical composition comprising a protein of the invention, and, optionally, comprising a suitable adjuvant or expedient, can be employed for this purpose.

The polypeptide of the invention is used for the preparation of a pharmaceutical composition for treating subjects allergic to the venom of an insect from the order Hymenoptera. The invention thus provides a method of treating subjects allergic to the venom of an insect from the order Hymenoptera, comprising administering a polypeptide of the invention to a subject having such an allergy.

Desensitization approaches are well known in the state of the art. In principle, repeated treatments of allergic individuals with suitable, normally progressively increased doses of allergen diverts the immune response to one dominated by T cells that favour the production of IgG and IgA antibodies over production of IgE antibodies. The IgG and IgA antibodies are thought to desensitize the subject by binding to the small amounts of allergen normally encountered, and preventing the allergen from binding to IgE. Desensitization to insect or bee venom is almost universally successful (Hunt et al 1978). Different protocols and time schedules can be used, from traditional protocols, rush protocols to ultrarush protocols (e.g., Schiavino et al 2004), all of which are incorporated herein by reference. The efficacy of such protocols can be evaluated by testing the adjustment of IgE and IgG (different isotypes) and/or IgA levels in the subject's blood or by challenging the subject in a controlled manner and determining the allergic response.

The Api m 3 polypeptide, a fragment, a derivative or an analog thereof is administered over a period of time in gradually increasing doses effective to reduce the allergic response of the individual to the protein allergen. Examples of routes of administration include parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., sublingual or via inhalation), transdermal (topical), and rectal administrations. The effective amount of the Api m 3 polypeptide, a fragment, a derivative and an analog thereof will vary according to factors such as the degree of sensitivity of the individual to Api m 3, the age, sex, and weight of the individual, and the ability of the fragment, derivative, or analog thereof to elicit an antigenic response in the individual. In one embodiment, the amount of Api m 3 polypeptide administered to an individual corresponds to the amount of Api m 3 in the venom of vespids that is injected into an individual by a sting. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily.

The polypeptide of the invention can be administered alone or combination with other allergens, e.g. other Hymenoptera venom proteins or fragments thereof. In particular, combinations with bee or Hymenoptera venom phospholipase A2, hyaluronidase, glucosidase and/or mellitin are suitable, as this therapy induces generation of IgG/IgA antibodies to several venom allergens and can thus lead to full protection. The identified bee allergens are shown in Table 2.

In a specific embodiment, the present invention features a method of modulating an immune response by administering T cell epitope-containing peptides of at least 9 amino acids corresponding to a consecutive amino acid sequence within Api m 3 to a subject in need thereof in an amount sufficient to inhibit an immune reaction by the subject against the Api m 3 polypeptide. If desired, T cell epitope-containing peptides of at least 9 amino acids corresponding to a consecutive amino acid sequence within one or more additional polypeptides, e.g., within a second, third, fourth, or more honeybee venom polypeptide or polypeptides can be comprised in such peptides. The additional honeybee venom polypeptides can include, e.g., the Api m 1 polypeptide (phospholipase A2), the Api m 2 polypeptide (hyaluronidase), the Api m 4 oligopeptide (mellitin), the Api m 5 polypeptide, (allergen C, dipeptidylpeptidase), and other glycosylated or non-glycosylated IgE-binding honeybee venom proteins, or analogs or derivatives thereof.

As used herein, a decrease or modification of the T cell response of a mammal sensitive to a protein allergen is defined as non-responsiveness or diminution in symptoms to the protein allergen in the mammal, as determined by standard clinical procedures (see e.g., Varney et al 1991). As referred to herein, a diminution in symptoms to an allergen includes any reduction in the allergic response of a mammal (such as a human) to the allergen following a treatment regimen with a polypeptide as described herein. This diminution in symptoms may be determined subjectively in humans (e.g., the patient feels more comfortable upon exposure to the allergen), or clinically, such as with a standard skin test.

The polypeptide of the invention can also be used for the preparation of a diagnostical composition for diagnosing or identifying subjects allergic to the venom of an insect from the order Hymenoptera. A method of diagnosing an allergy to venom of an insect from the order Hymenoptera is thus provided, comprising the steps of

-   -   a) contacting a subject with a polypeptide of the invention and     -   b) detecting an allergic reaction, wherein detecting an allergic         reaction indicates said allergy.

In vivo tests for diagnosis of an allergy can easily be adapted to the polypeptide of the invention. Typically, a suitable amount of allergen is injected subcutaneously into a subject's limb, and, after a certain amount of time, the degree of localised inflammation in comparison to controls is determined (skin prick test). Such tests are well known in the art (Hamilton 2002, Poulsen 2001, Schmid-Grendelmeier 2001, Williams et al 1999, Barbee et al 1976).

An allergy to the venom of an insect from the order Hymenoptera can also be diagnosed by an in vitro method comprising the steps of

-   -   a) in vitro contacting a blood sample from a subject with a         polypeptide of the invention and     -   b) detecting binding of IgE antibodies to the polypeptide,         wherein detecting IgE antibodies binding to the polypeptide         indicates said allergy.

Binding of IgE antibodies to the polypeptide can, e.g., be detected in an ELISA or by an in vitro release assay employing stripped mast cells and measuring the amount of released mediator, e.g., histamine. To determine specific binding, the results are compared with a specificity control, e.g., with an unrelated antibody. The diagnostic tests can in parallel be carried out to determine the levels of specific IgG (in particular IgG1 and/or IgG4) and/or IgA. For this, an ELISA with specific secondary antibodies recognising the different isotypes can be employed. Parallel testing is particularly useful for following and evaluating a course of specific immunotherapy.

In another embodiment, the present invention provides in vitro diagnostic assays on peripheral blood lymphocytes useful for obtaining information on Api m 3-specific T cell responses, the phenotype of the T cell response, and preferably the T cell epitope(s) of Api m 3 involved in T cell responses. The immunodominant epitope(s) and the epitope(s) involved in IgE isotype class switch events can be detected, if they are not identical. In particular, the T cell epiotope(s) of Api m 3 that stimulate proliferation and/or lymphokine secretion of T cells of a phenotype associated with IgE isotype class switching events can be identified for a specific individual, or for a class of individuals who share MHC haplotype or a predominant T cell receptor variable region expression, or both.

For the therapeutic and diagnostic uses and methods, it is preferred to employ the fusion polypeptides of the invention, non-glycosylated proteins or polypeptides that are capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera, and comprise at least 5, preferably at least 6, 7, 8, 9, 10, 15, 20 or more amino acids of a polypeptide more than 70%, more than 80% or more than 90% homologous or identical to a polypeptide selected from the group consisting of amino acid 26 to 99, 116 to 145, 153 to 158, 185 to 223, 232 to 276 or 362 to 373 of the polypeptide shown in SEQ ID NO: 2. Alternatively, the employed polypeptides are capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera, and comprise at least 5, preferably at least 6, 7, 8, 9, 10, 15, 20 or more amino acids of a polypeptide more than 70%, more than 80% or more than 90% homologous or identical to the polypeptide shown in SEQ ID NO: 2, except for the polypeptides from the group consisting of amino acids 1 to 34, 63 to 80, 100 to 115, 142 to 149, 168 to 176, 224-239 and 258 to 343 of the polypeptide shown in SEQ ID NO: 2 or except for the polypeptides shown in FIG. 5. Additionally, such polypeptides consisting of amino acids 1 to 25, 146 to 152, 159 to 164, 168 to 184, 224 to 231, and 277 to 361 can be used.

In one embodiment, the Api m 3 polypeptide, fragments, derivatives and/or analogs thereof, are incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the Api m 3 polypeptide, fragments, derivatives or analogs thereof, and a pharmaceutically acceptable carrier. As used herein, a ‘pharmaceutically acceptable carrier’ is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and adsorption delaying systems, and the like, compatible with the active compound and pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active compounds can also be incorporated into the composition. As used herein, the phrases ‘pharmaceutical composition’ and ‘medicament’ are interchangeable.

In another embodiment, the pharmaceutical composition includes an additional polypeptide, e.g., a second, third, fourth, or more honeybee venom polypeptide or polypeptides. The additional honeybee venom polypeptides can include, e.g., the Api m 1 polypeptide (phospholipase A2), the Api m 2 polypeptide (hyaluronidase), the Api m 4 oligopeptide (mellitin), the Api m 5 polypeptide, (allergen C, dipeptidylpeptidase), and other glycosylated or non-glycosylated IgE-binding honeybee venom proteins, or analogs or derivatives thereof.

In another embodiment, the present invention features a pharmaceutical composition comprising Api m 3 polypeptide fragments of the invention, preferably between 20-150 amino acids in length, wherein each fragment contains one or more B cell epitopes and one or more T cell epitopes, and a pharmaceutically acceptable carrier.

In another embodiment, the pharmaceutical composition includes polypeptide fragments derived from an additional polypeptide, e.g., a second, third, fourth, or more honeybee venom polypeptides or oligopeptides including, but not limited to, the Api m 1 polypeptide (phospholipase A2), the Api m 2 polypeptide (hyaluronidase), the Api m 4 oligopeptide (mellitin), the Api m 5 polypeptide, (allergen C, dipeptidylpeptidase), and other glycosylated or non-glycosylated IgE-binding honeybee venom proteins, or analogs or derivatives thereof.

In another embodiment, the pharmaceutical composition includes Api m 3 polypeptide fragments of the invention, fused to polypeptide fragments derived from an additional polypeptide, e.g., a second, third, fourth, or more honeybee venom polypeptides or oligopeptides including, but not limited to, the Api m 1 polypeptide (phospholipase A2), the Api m 2 polypeptide (hyaluronidase), the Api m 4 oligopeptide (mellitin), the Api m 5 polypeptide, (allergen C, dipeptidylpeptidase), and other glycosylated or non-glycosylated IgE-binding honeybee venom proteins, or analogs or derivatives thereof.

In another embodiment, the present invention features a pharmaceutical composition comprising T cell epitope containing peptides of at least 9 amino acids corresponding to a consecutive amino acid sequence within Api m 3 wherein the peptides are capable of stimulating T cells of subjects allergic to Api m 3. In a preferred embodiment, the composition comprises a set of T cell epitope-containing peptides capable of stimulating T cells of the great majority of subjects allergic to Api m 3.

In another embodiment, the pharmaceutical composition includes T cell epitope-containing peptides of at least 9 amino acids corresponding to a consecutive amino acid sequence within an additional polypeptide, e.g., a second, third, fourth, or more honeybee venom polypeptides or oligopeptides including, but not limited to, the Api m 1 polypeptide (phospholipase A2), the Api m 2 polypeptide (hyaluronidase), the Api m 4 oligopeptide (mellitin), the Api m 5 polypeptide, (allergen C, dipeptidylpeptidase), and other glycosylated or non-glycosylated IgE-binding honeybee venom proteins, or analogs or derivatives thereof.

Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates or phosphates and agents for the adjustment of toxicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. The composition should be fluid to the extent that easy syringability exists. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case dispersion and by use of surfactants. The composition should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimoseral, and the like. Delayed absorption of the injectable compositions can be achieved by including in the composition an agent such as aluminum monostearate and gelatin. In all cases, the composition must be sterile. Sterile injectable solutions can be prepared by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatine capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as mouthwash, wherein the active compound in the fluid carrier is swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatine; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavouring agent such as peppermint, methyl salicylate, or orange flavouring.

For administration by inhalation, the active compounds are delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g. a gas such as carbon dioxide, or a nebulizer.

For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include for transmucosal administration, for example; detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. Suppositories can be prepared using conventional suppository base such as cocoa butter or other glycerides. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. For rectal delivery the compounds can also be prepared in the form of retention enemas.

In a further embodiment, the active compounds are prepared with carriers that will protect the active compounds against rapid elimination from the body, such as controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polyacetic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

For oral and parenteral applications it is advantageous to formulate the compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated. Each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the included pharmaceutical carrier. The specification for the dosage unit forms of the invention are dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

The present invention also relates to a method of diagnosing an allergy to venom of an insect from the order Hymenoptera, comprising the steps of

-   -   a) performing the method of producing a polypeptide encoded by         the nucleic acid of the invention, wherein the host cell         comprising the expression vector of the invention is cultured         under appropriate conditions for expression of said polypeptide,         and wherein said polypeptide is purified,     -   b) contacting the polypeptide obtained by the method of step a)         in vitro with a blood sample,     -   c) and detecting binding of IgE antibodies to the polypeptide,         wherein detecting IgE antibodies binding to the polypeptide         indicates said allergy.

Furthermore, a method of diagnosing an allergy to venom of an insect from the order Hymenoptera is provided, comprising the steps of

-   -   a) performing the method of producing a polypeptide encoded by         the nucleic acid of the invention, wherein the host cell         comprising the expression vector of the invention is cultured         under appropriate conditions for expression of said polypeptide,         and wherein said polypeptide is purified,     -   b) contacting a subject with the polypeptide obtained by the         method of step a) and detecting an allergic reaction, and     -   c) detecting an allergic reaction, which is indicative of the         allergy.

The invention also provides a method of preparing a composition for diagnosing an allergy to venom of an insect from the order Hymenoptera comprising the step of producing a polypeptide encoded by the nucleic acid of the invention, wherein the host cell comprising the expression vector of the invention is cultured under appropriate conditions for expression of said polypeptide and said polypeptide is purified and can be used as such for diagnosis. Optionally, the polypeptide is further formulated with stabilizers, such as a neutral protein (e.g., BSA) or detergents to give said composition.

In another embodiment, the invention teaches a method of preparing a composition for treating subjects allergic to the venom of an insect from the order Hymenoptera, comprising the step of performing the method of producing a polypeptide encoded by the nucleic acid of the invention, wherein the host cell comprising the expression vector of the invention is cultured under appropriate conditions for expression of said polypeptide and said polypeptide is purified and can be used as such for therapy. Optionally, the polypeptide is further formulated with appropriate excipient and/or carriers in order to provide said composition. Correspondingly, a method of treating subjects allergic to the venom of an insect from the order Hymenoptera is disclosed, comprising the steps of

-   -   a) performing the method of producing a polypeptide encoded by         the nucleic acid of the invention, wherein the host cell         comprising the expression vector of the invention is cultured         under appropriate conditions for expression of said polypeptide         and said polypeptide is purified, and     -   b) administering the polypeptide obtained by the method of         step a) to a subject having such an allergy.

The present invention thus for the first time satisfies the need for a recombinantly produced Hymenoptera venom acid phosphatase or the cDNA encoding this polypeptide, which can be used for diagnostic and therapeutic applications.

EXAMPLES Example 1 Cloning of cDNA

1.1 Total RNA Isolation

Total RNA was isolated from the separated stinger of a honey bee with attached venom sack and additional glands. The isolation of total RNA was performed using a kit according to the manual (peqGold TriFast™, peqlab Biotechnologie GmbH, Erlangen, Germany). The organ was weighed and homogenised in a solution containing guanidiniumisothiocyanate and phenol. Phase separation was induced by addition of chloroform. The aqueous phase was separated after centrifugation, and the containing RNA precipitated with isopropyl alcohol. After washing with diluted ethanol the RNA was dissolved in RNase-free sterile water and used directly in RT-PCR experiments. To prepare RNase-free sterile water cell-culture suitable water was treated with 0.1% (v/v) dimethylpyrocarbonate (DEPC) overnight, and then autoclaved for 20 minutes to destroy DEPC by causing hydrolysis of DEPC.

1.2 cDNA First Strand Synthesis

Reverse transcriptase was used to synthesise first strand cDNA from the isolated RNA. For this 5 μl of total bee RNA was mixed with 2 μl (20 pmol) oligonucleotide primer and 4 μl DEPC water. An universal oligo-dT of 20 base pair length was used for the purpose of transcribing the poly-adenylated portion of mRNA in the total RNA sample. The reaction mix was incubated at 70° C. for 5 minutes to break secondary structures. After this, the reaction was chilled on ice. Subsequently, 1.5 μl DEPC water, 4 μl 5× reaction buffer, 2 μl dNTP mix (10 mM), and 0.5 μl RNaseOut™ recombinant ribonuclease inhibitor (Invitrogen GmbH, Karlsruhe, Germany) were added. The reaction mix was incubated at 37° C. for 5 minutes. Then 1 μl (200 units) RevertAid™ M-MuLV Reverse Transcriptase (RT, Fermentas GmbH, St. Leon-Rot, Germany) was added and the reaction was incubated at 42° C. for 60 minutes. After this the reaction was stopped by heating to 70° C. for 10 minutes and chilled on ice.

1.3 RT-PCR

First strand cDNA from bee venom gland tissue was used as template for PCR amplification of Api m3 DNA sequences.

Known peptide fragments, public databases and bioinformatics were used to design the specific primers for Api m3. These primers have been designed to allow 5′-end blunt subcloning for native N-terminal expression and 3′-end directed Sac II restriction site subcloning. The nucleotide sequences of the oligonucleotides are:

Api m3 for, 21 mer, blunt end (SEQ ID NO: 3): 5′-GAA CTT AAA CAA ATA AAT GTG Api m3 back 32 mer, Sac II site (SEQ ID NO: 4): 5′-AAC CGC GGT TAC TTA CTT ATT CTC AGT ACC CG.

The PCR reaction contained 41 μl DEPC water, 5 μl 10× complete Pfu PCR buffer, 1 μl Api m3 for primer (100 pmol), 1 μl Api m3 back primer (100 pmol), 1 μl dNTP mix (10 mM), 0.5 μl bee venom gland tissue cDNA, and 0.5 μl recombinant Pfu DNA polymerase (Fermentas GmbH, St. Leon-Rot, Germany), to give a total reaction volume of 50 μl.

The PCR temperature program for amplification was:

Step 1: 96° C., 1 minute

Step 2: 95° C., 30 seconds

Step 3: 55° C., 30 seconds

Step 4: 72° C., 2 minutes

Repeat steps 2-4×29 times

Step 5: 72° C., 10 minutes

Step 6: 4° C., until end

Part of the PCR reaction was run on a 1% agarose (peqGOLD universal agarose, peqlab GmbH, Erlangen, Germany) gel in 0.5×TAE buffer and amplified DNA products visualised with ethidium bromide and UV illumination. A band at the expected size was visible.

1.4 Subcloning for Sequencing

DNA from the PCR reaction was isolated using the QIAEX II gel extraction kit (Qiagen GmbH, Hilden, Germany). Subcloning for sequencing was done using the TOPO TA Cloning® Kit (Invitrogen GmbH, Karlsruhe, Germany) with pCR®2.1-TOPO® vector according to the manual. Due to use of Pfu DNA polymerase an initial TA-elongation reaction step with AGS Gold Taq DNA Polymerase (AGS Hybaid, Heidelberg) was introduced. The ligated DNA was transformed into E. coli of the strain TG1 by electroporation (2 mm cuvettes, EasyJect+, Hybaid, Heidelberg, Germany) and selected on ampicillin agar plates.

1.5 Sequencing

The sequencing reaction was done with BigDye® Terminator Cycle Sequencing Kit from ABI (Applied Biosystems Applera Deutschland GmbH, Darmstadt, Germany) according to the manual. 25 cycles were run with a 30 seconds denaturation step at 96° C., 15 seconds annealing step at 50° C., and 4 minutes elongation step at 57° C. Sequencing primer were:

M13/Uni for (SEQ ID NO: 5): 5′-GTA AAA CGA CGG CCA GTG CCA A M13/Uni rev (SEQ ID NO: 6): 5′-CAG GAA ACA GCT ATG ACC ATG A

The resulting sequence is shown in FIG. 1.

Example 2 Construction of Expression Vector

2.1 Modification of the Insect Expression Vector

For expression of recombinant Api m 3 with potential for native folding and posttranslational modification, the expression in insect cells was chosen. The expression vector pIB/V5-His. (Invitrogen GmbH, Karlsruhe, Germany) was modified to facilitate isolation and purification. A melittin signal sequence for secretion of the recombinant protein was added and the Kozak sequence was optimised for higher expression rates in insect cells. The melittin signal sequence was amplified from total bee RNA, synthesised as described above, using the primers:

melt leader for (SEQ ID NO: 7): 5′-GGA AAG CTT TCC GCC ATG GCG AAA TTC TTA GTC melt leader back (SEQ ID NO: 8): 5′-CGG GAT CCC GCA TAG ATG TAA GAA ATG.

Underlined are the Hind III and, respectively, BamH I restriction sites in the corresponding primer. The sequence containing the 10× histidine-tag and factor Xa cleavage site has been cloned between the BamH I and EcoR V site of the parent vector. As first template, a tag containing vector was used with the following primers:

10xHis for (SEQ ID NO: 9): 5′-CTG AAT AGC GCC GGA TCC GAC CAT 10xHis back (SEQ ID NO: 10): 5′-CCC TCT AGA CTC GAG CCA ATG ATG

Underlined are the bases for the introduction of the BamH I restriction site. The resulting fragment was used as second template and further modified to contain a EcoR V site at the 3′-end by use of overlapping primers and PCR extension of the sequence (splice-overlap-extension, SOE). The extension primer used was:

SOE Xa (SEQ ID NO: 11): 5′-GGG ATA TCC CTT CCC TCG ATC CCT CTA GAC TC

Underlined is the newly introduced EcoR V restriction site for cloning and generation of the expression vector construct. For all PCR steps Pfu DNA polymerase (Fermentas GmbH, St. Leon-Rot, Germany) was used with standard reaction conditions. The annealing temperature was 55° C. for the 10× Histidin fragment amplification and 45° C. for the SOE reaction.

2.2 Re-PCR and Subcloning

After sequencing of selected subcloned cDNA clones and verification of the sequence, the clone was used for secondary amplification with Pfu DNA polymerase. The PCR product was subcloned into the EcoR V/Sac II digested expression vector after restriction digest with Sac II.

2.3 Modification of the Bacterial Expression Vector

The verified mammalian expression vector pIB/MeI opt-H10 was used as template for the construction of insert for subcloning into the prokaryotic expression vector pET26(+) (Novagen). The PCR program was done according to the temperature gradient given in 1.3. Pfu polymerase was used with the primers:

(SEQ ID NO: 12) Api 3 for pro-his AGAATTTCATATGAAATTCTTAGTCAACG (SEQ ID NO: 13) Api 3 back pro AAGAGCTCTTACTTACTTATTCTCAG

The amplicon was digested with Sac I and Nde I. The partly digested fragment of correct size was isolated and ligated into the pre-digested vector.

Example 3 Expression of Recombinant Protein

3.1 Transfection

HighFive insect cell (Invitrogen GmbH, Karlsruhe, Germany) were used as hosts for the recombinant expression of Api m 3. DNA was purified from bacterial cultures using the E.Z.N.A. Plasmid Miniprep Kit II (peqlab GmbH, Erlangen, Germany) according to the manual. For transfection of purified DNA into cells the reagent Cellfectin® (Invitrogen GmbH, Karlsruhe, Germany) was used according to the manual.

3.2 Transformation

Vectors have been transformed into prokaryotic cell by electroporation. Cells have been prepared by standard procedures. Electroporation was done with an EasyJecT+ instrument (EquiBio, Maidstone, UK) with standard settings according to the manual of the manufacturer.

3.3 Isolation of Recombinant Protein

The protein was purified according to standard procedures.

In brief, prokaryotic cells were disrupted by sonication. Cell membranes etc. were sedimented by ultracentrifugation. The His-tagged protein was then purified from the extract by Ni²⁺ affinity chromatography following the manufacturer's recommendations (e.g., His Trap™ HP Kit, Amersham Biosciences). Purification was controlled by SDS-PAGE. In the case of eukaryotic expression the supernatant medium was collected from confluent stably transfected insect cell expression cultures. The supernatant was adjusted to pH 7.8 and centrifuged at 4000×g for 5 minutes. Aliquots of 10-20 ml medium were applied to a nickel-chelating affinity matrix (NTA-agarose, Qiagen). The column was washed with 10 ml NTA-binding buffer (50 mM sodium phosphate, pH 7.6, 500 mM NaCl) and pre-eluted with NTA-binding buffer containing 20 mM imidazole. The recombinant protein was finally eluted from the matrix with 10 ml NTA-binding buffer containing 400 mM imidazole. Purification was controlled by SDS-PAGE and silver staining of protein.

Example 4 Analysis of Recombinant Api m 3

4.1 Sequence Alignment and Motif Analysis

Sequence databases were screened with BLAST algorithms for related sequences of the cloned Api m 3 in other organisms. Sequence alignment was performed with four homologous sequences found in the organisms Drosophila melanogaster and Drosophila subobscura coding for acid phosphatases. The sequences show significant homologies. The highest homology with 35% is found for Acph-1 from D. melanogaster. Amino acids necessary for acid phosphatase activity (RHGXRXP motif) are highly conserved in the sequence. In addition, four potential N-glycosylation sites (NXS/T motif) have been identified.

4.2 Tryptic Fragment Prediction

To verify the cloned sequence matches the expressed recombinant protein a prediction of tryptic fragments was done based on the nucleic acid sequence. The purified protein was digested with sequence grade Trypsin (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) according to the instructions of the manufacturer and the resulting peptide fragments were analyzed by MALDI-TOF spectrometry using standard protocols. The predicted fragments matched the data acquired by MALDI-TOF and therefore verified the identity of the recombinant protein.

4.3 Enzymatic Activity Assay

Enzymatic activity of the recombinant enzyme was confirmed according to a described method (Barboni et al 1987).

Example 5 Immunoreactivity of Recombinant Api m 3

Recombinant Api m 3 isolated from stably transfected insect cells was used in an immunoprinting experiment with serum from honey bee venom allergic patients to evaluate IgE reactivity. Diluted honey bee venom and purified recombinant Api m 3 were examined in the same experiment. Proteins were separated on 10% SDS-PAGE gels under reducing conditions. Transfer to nitrocellulose membrane (Protran, Schleicher & Schuell BioScience GmbH, Dassel, Germany) and subsequent immunostaining for sIgE reactive allergens was done using a kit according to the manual (AlaBLOT kit, DPC Biermann GmbH, Bad Nauheim, Germany) showing the immunoreactivity of recombinant Api m 3.

Example 6 Patient Screening with Recombinant Api m 3

Immunoreactivity assays with sera from individual patients To detect specific IgE immunoreactivity of human sera with purified recombinant Api m 3, ELISA plates (NUNC GmbH & Co. KG, Wiesbaden, Germany) were coated with 100 μl of purified recombinant Api m 3 (1 μg/ml) or, as a positive control, purified natural Api m 1 (1 μg/ml) (Latoxan, Valence, France) at 4° C. overnight. For all reaction steps, an ELISA buffer reagent set was used according to the manual (BD Biosciences, Heidelberg, Germany). Appropriate dilutions (1:2; 1:5; 1:10) of the sera were made in assay diluent. Bound IgE was detected with a biotinylated mouse anti-human IgE (BD Biosciences) together with horseradish peroxidase-conjugated avidin, both diluted 1:250 in assay diluent. Color was developed with 100 μl substrate solution per well for 30 minutes in the dark. Finally, 50 μl stop solution were added and plates were read at 450/570 nm. For quality control of the assay, an 8-point human IgE standard curve was run on each plate using murine anti-IgE (10 μg/ml) as capture antibody and human myeloma IgE (Calbiochem-Merck, Darmstadt, Germany) over a concentration range of 31.25 to 4,000 pg/ml (100 μl per well, diluted in assay diluent). Secondary antibody and detection system for total IgE were identical to the one described above for the detection of Api m 1/rApi m 3 sIgE. It could be shown that approximately 37.5% (15/40) of the patient sera that were characterized by a positive sIgE test to honeybee venom had detectable sIgE to recombinant Api m 3. Of 19 patients lacking serologic reactivity to honeybee venom (sIgE <0.35 kU/L), 10 patients were highly sensitized to Vespula spp. venom but non-reactive towards honeybee venom (SIgE≧50 kU/L, FIG. 6B) and 9 were individuals lacking serologic IgE reactivity to both hymenoptera venoms (sIgE <0.35 kU/L to both, vespid and honeybee venom). Only one serum out of the 19 sera lacking serologic reactivity to honeybee venom showed reactivity with recombinant Api m 3. This patient had a clearcut positive sIgE result in the recombinant Api m 3 ELISA. He reported to the allergy service with a history of a severe anaphylactic reaction after a hymenoptera sting. The offending insect was not identified by the patient. Despite a negative “classical” serologic result and a negative intradermal skin test, the patient was finally classified as an honey bee venom allergic patient. It can be assumed that he reacts strongly to native Api m 3 with is likely to be underrepresented in clinical test kits and therefore his allergy was not noticed.

Example 7

Improved differentiation between sera binding to different epitopes based on recombinantly expressed Api m 3

a) Api m3 Expressed in Bacterial Cells:

The different structural features of recombinant Api m 3 expressed in bacterial cells are documented by following experiments (experimental conditions as described in Example 6):

As shown in FIG. 1, native Api m 3 purified according to document D1 is recognized by IgE in 6 of 9 (66%) sera from patients with honeybee venom allergy. This result is in excellent accordance with data published by Kemeny et al. (1983). Using purified native Api m 3, Kemeney and coworkers demonstrated serum IgE to Api m 3 in 60% of the sera from patients with honeybee venom allergy.

In contrast, recombinant Api m 3 expressed in bacterial cells (E. coli) is recognized by IgE in a significantly lower number of sera from patients with honeybee venom allergy. When expressed as fusion protein with bacterial maltose binding protein (MBP), Api m 3 is recognized by IgE in 3 of 9 (33%) sera from patients with honeybee venom allergy (see FIG. 14A). When expressed as fusion protein with eukaryotic glutathion-S-transferase (GST), Api m 3 is recognized by IgE in only 2 of 9 (22%) sera from patients with honeybee venom allergy (see FIG. 14B).

b) Api m3 Expressed in Insect Cells:

Recombinant Api m 3 molecules expressed in insect cells (HighFive cells or SF9 cells) are glycosylated, but the glycosylation pattern provided by both insect cell lines to Api m 3, exhibits significant differences. As a result, both Api m 3 molecules are recognized by IgE in different sera from patients with honeybee venom allergy.

The profound effects of different glycosylation patterns of Api m 3 expressed in different insect cells, on the binding of IgE antibodies are documented by following experiments:

Api m 3 expressed in HighFive insect cells is recognized by IgE in 6 of 9 (66%) sera from patients with honeybee venom allergy and, however, partly by different sera than native Api m 3 (see FIG. 15A). Api m 3 expressed in SF9 insect cells is recognized by IgE in only 3 of 9 (33%) sera from patients with honeybee venom allergy (see FIG. 15B).

Furthermore, FIG. 16 demonstrates that both molecules are recognized to a different extent by IgE in sera from patients allergic to both honeybee and wasp venom, which allows for an improved evaluation of carbohydrate based cross-reactivity of IgE antibodies. The data in FIG. 16 show that recombinant Api m 3 produced in HighFive insect cells is recognized by IgE in 19 of 23 (82%) sera from patients allergic to both honeybee and wasp venom. Ten of these sera contain IgE that is highly reactive with Api m 3 produced in HighFive insect cells. It should be stressed that the sera tested in FIG. 16 are obtained from patients allergic to both honeybee and wasp venom and, therefore, cannot be compared to those sera tested in FIGS. 13-15 which are obtained from patients allergic only to honeybee venom.

In summary, the structural features of recombinant Api m 3 expressed in E. coli and insect cells differ significantly from those of native Api m 3.

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TABLE 1 Bee venom components % weight of Component type name dry mass Proteins Phospholipase A2 (Api m 1) 10-12 Hyaluronidase (Api m 2) 1-3 Phosphatase, Glucosidase 1-2 Peptides Melittin (Api m 4) 50-55 Secapin, MCD-peptide 1.5-4   Tertiapamin, Apamin, Procamin 2-5 Other small peptides 13-15 Biogene amines Histamine 0.5-2   Dopamine 0.2-1   Norepinephrine 0.1-0.5 Sugars (Glucose, Fructose) 2 Phospholipids 5 Amino acids — Volatile Pheromones 4-8 substances Minerals 3-4

TABLE 2 Identified bee allergens Allergen Common name Size (processed) Weight SwissProt Reference Api m 1 Phospholipase A2 134 aa 15.2 kDa P00630 Kuchler et al 1989 Api m 2 Hyaluronidase 349 aa 40.7 kDa Q08169 Gmachl and Kreil 1993 Api m 3 Acid Phosphatase nd   45 kDa — Barboni et al 1987 Api m 4 Melittin  26 aa  2.8 kDa P01501 Vlasak et al 1983 Api m 5 Allergen C nd  105 kDa — Hoffman et al 1977 Api m 6 —  71 aa  7.5 kDa P83563 Kettner et al 2001

TABLE 3 NetMHCII 1.0 predicted T cell epitopes in Api m 3 (only strong and weak binders) Peptide Start No.* End No.* Start No.** End No.** Length Allele No. nucleic acid nucleic acid amino acid amino acid amino acid DRB1*0101 1 307 351 103 117 15 2 310 354 104 118 15 3 313 357 105 119 15 4 316 360 106 120 15 5 319 363 107 121 15 6 523 567 175 189 15 7 526 570 176 190 15 8 514 558 172 186 15 9 517 561 173 187 15 10 520 564 174 188 15 11 655 699 219 233 15 12 661 705 221 235 15 13 658 702 220 234 15 14 652 696 218 232 15 15 664 708 222 236 15 16 889 933 297 311 15 17 880 924 294 308 15 18 883 927 295 309 15 19 886 930 296 310 15 20 892 936 298 312 15 21 532 576 178 192 15 22 529 573 177 191 15 23 322 366 108 122 15 24 325 369 109 123 15 25 667 711 223 237 15 26 670 714 224 238 15 27 622 666 208 222 15 28 535 579 179 193 15 29 184 228 62 76 15 30 190 234 64 78 15 31 187 231 63 77 15 32 193 237 65 79 15 33 247 291 83 97 15 34 253 297 85 99 15 35 895 939 299 313 15 36 250 294 84 98 15 37 625 669 209 223 15 38 628 672 210 224 15 39 244 288 82 96 15 40 181 225 61 75 15 41 241 285 81 95 15 42 898 942 300 314 15 43 1066 1110 356 370 15 44 619 663 207 221 15 45 1069 1113 357 371 15 46 616 660 206 220 15 47 1063 1107 355 369 15 48 1060 1104 354 368 15 49 256 300 86 100 15 50 793 837 265 279 15 51 259 303 87 101 15 52 541 585 181 195 15 53 754 798 252 266 15 54 796 840 266 280 15 55 751 795 251 265 15 56 775 819 259 273 15 57 784 828 262 276 15 58 802 846 268 282 15 59 778 822 260 274 15 60 745 789 249 263 15 61 748 792 250 264 15 62 1072 1116 358 372 15 63 787 831 263 277 15 64 781 825 261 275 15 65 799 843 267 281 15 66 805 849 269 283 15 67 742 786 248 262 15 68 235 279 79 93 15 69 238 282 80 94 15 70 196 240 66 80 15 71 631 675 211 225 15 72 538 582 180 194 15 73 634 678 212 226 15 74 1075 1119 359 373 15 75 199 243 67 81 15 76 610 654 204 218 15 77 547 591 183 197 15 78 613 657 205 219 15 79 550 594 184 198 15 80 1057 1101 353 367 15 81 553 597 185 199 15 82 544 588 182 196 15 83 301 345 101 115 15 84 304 348 102 116 15 85 262 306 88 102 15 86 385 429 129 143 15 87 388 432 130 144 15 88 673 717 225 239 15 89 382 426 128 142 15 90 379 423 127 141 15 91 685 729 229 243 15 92 265 309 89 103 15 93 679 723 227 241 15 94 808 852 270 284 15 95 562 606 188 202 15 96 760 804 254 268 15 97 757 801 253 267 15 98 682 726 228 242 15 99 688 732 230 244 15 100 91 135 31 45 15 101 232 276 78 92 15 102 94 138 32 46 15 103 676 720 226 240 15 104 556 600 186 200 15 105 790 834 264 278 15 106 229 273 77 91 15 107 877 921 293 307 15 108 226 270 76 90 15 109 1021 1065 341 355 15 110 394 438 132 146 15 111 1015 1059 339 353 15 112 1018 1062 340 354 15 113 1024 1068 342 356 15 114 811 855 271 285 15 115 511 555 171 185 15 116 391 435 131 145 15 117 691 735 231 245 15 118 1012 1056 338 352 15 119 508 552 170 184 15 120 958 1002 320 334 15 121 568 612 190 204 15 122 565 609 189 203 15 123 574 618 192 206 15 124 571 615 191 205 15 125 955 999 319 333 15 126 82 126 28 42 15 127 85 129 29 43 15 128 874 918 292 306 15 129 88 132 30 44 15 130 505 549 169 183 15 DRB1*0401 131 616 660 206 220 15 132 622 666 208 222 15 133 619 663 207 221 15 134 613 657 205 219 15 135 610 654 204 218 15 136 625 669 209 223 15 137 628 672 210 224 15 138 190 234 64 78 15 139 187 231 63 77 15 140 193 237 65 79 15 141 181 225 61 75 15 142 184 228 62 76 15 143 745 789 249 263 15 144 742 786 248 262 15 145 748 792 250 264 15 146 754 798 252 266 15 147 196 240 66 80 15 148 199 243 67 81 15 149 751 795 251 265 15 150 880 924 294 308 15 151 883 927 295 309 15 152 514 558 172 186 15 153 886 930 296 310 15 154 889 933 297 311 15 155 517 561 173 187 15 156 511 555 171 185 15 157 520 564 174 188 15 158 892 936 298 312 15 DRB1*0404 159 703 747 235 249 15 160 697 741 233 247 15 161 691 735 231 245 15 162 700 744 234 248 15 163 694 738 232 246 15 164 667 711 223 237 15 165 679 723 227 241 15 166 676 720 226 240 15 167 670 714 224 238 15 168 673 717 225 239 15 169 706 750 236 250 15 170 709 753 237 251 15 171 562 606 188 202 15 172 685 729 229 243 15 173 682 726 228 242 15 174 565 609 189 203 15 175 568 612 190 204 15 176 841 885 281 295 15 177 838 882 280 294 15 DRB1*0405 178 301 345 101 115 15 179 556 600 186 200 15 180 553 597 185 199 15 181 967 1011 323 337 15 182 289 333 97 111 15 183 292 336 98 112 15 184 307 351 103 117 15 185 304 348 102 116 15 186 559 603 187 201 15 187 970 1014 324 338 15 188 973 1017 325 339 15 189 562 606 188 202 15 190 76 120 26 40 15 191 73 117 25 39 15 192 475 519 159 173 15 193 514 558 172 186 15 194 79 123 27 41 15 195 82 126 28 42 15 196 976 1020 326 340 15 197 295 339 99 113 15 198 478 522 160 174 15 199 517 561 173 187 15 200 979 1023 327 341 15 201 511 555 171 185 15 202 298 342 100 114 15 203 508 552 170 184 15 204 565 609 189 203 15 205 310 354 104 118 15 206 481 525 161 175 15 207 484 528 162 176 15 208 883 927 295 309 15 209 313 357 105 119 15 210 880 924 294 308 15 211 487 531 163 177 15 212 70 114 24 38 15 213 394 438 132 146 15 214 397 441 133 147 15 215 886 930 296 310 15 216 391 435 131 145 15 217 889 933 297 311 15 218 385 429 129 143 15 219 388 432 130 144 15 220 625 669 209 223 15 221 505 549 169 183 15 DRB1*0701 222 622 666 208 222 15 223 625 669 209 223 15 224 628 672 210 224 15 225 619 663 207 221 15 226 616 660 206 220 15 227 631 675 211 225 15 228 634 678 212 226 15 229 553 597 185 199 15 230 316 360 106 120 15 231 313 357 105 119 15 232 556 600 186 200 15 233 319 363 107 121 15 234 559 603 187 201 15 235 598 642 200 214 15 236 601 645 201 215 15 237 604 648 202 216 15 238 595 639 199 213 15 239 562 606 188 202 15 DRB1*0901 240 514 558 172 186 15 241 622 666 208 222 15 242 517 561 173 187 15 243 628 672 210 224 15 244 625 669 209 223 15 245 511 555 171 185 15 246 814 858 272 286 15 247 817 861 273 287 15 DRB1*1101 248 514 558 172 186 15 249 517 561 173 187 15 250 520 564 174 188 15 251 523 567 175 189 15 252 526 570 176 190 15 253 565 609 189 203 15 254 562 606 188 202 15 255 568 612 190 204 15 256 574 618 192 206 15 257 571 615 191 205 15 DRB1*1302 258 622 666 208 222 15 259 625 669 209 223 15 260 628 672 210 224 15 261 619 663 207 221 15 262 616 660 206 220 15 263 631 675 211 225 15 264 1066 1110 356 370 15 265 1069 1113 357 371 15 266 634 678 212 226 15 267 1063 1107 355 369 15 268 1072 1116 358 372 15 269 1075 1119 359 373 15 270 1060 1104 354 368 15 271 610 654 204 218 15 272 613 657 205 219 15 273 247 291 83 97 15 274 253 297 85 99 15 275 250 294 84 98 15 276 964 1008 322 336 15 277 259 303 87 101 15 278 568 612 190 204 15 279 256 300 86 100 15 280 562 606 188 202 15 281 571 615 191 205 15 282 565 609 189 203 15 283 1057 1101 353 367 15 284 574 618 192 206 15 285 967 1011 323 337 15 286 679 723 227 241 15 287 970 1014 324 338 15 288 682 726 228 242 15 289 958 1002 320 334 15 290 637 681 213 227 15 291 676 720 226 240 15 292 961 1005 321 335 15 293 871 915 291 305 15 294 955 999 319 333 15 295 874 918 292 306 15 296 973 1017 325 339 15 297 670 714 224 238 15 298 673 717 225 239 15 299 877 921 293 307 15 300 106 150 36 50 15 301 103 147 35 49 15 DRB1*1501 302 880 924 294 308 15 303 883 927 295 309 15 304 886 930 296 310 15 305 889 933 297 311 15 306 892 936 298 312 15 307 874 918 292 306 15 308 877 921 293 307 15 309 253 297 85 99 15 DRB4*0101 310 553 597 185 199 15 311 556 600 186 200 15 312 559 603 187 201 15 313 565 609 189 203 15 314 562 606 188 202 15 315 334 378 112 126 15 316 331 375 111 125 15 317 337 381 113 127 15 318 340 384 114 128 15 319 343 387 115 129 15 320 568 612 190 204 15 321 571 615 191 205 15 322 256 300 86 100 15 323 262 306 88 102 15 324 259 303 87 101 15 325 265 309 89 103 15 326 784 828 262 276 15 327 787 831 263 277 15 328 793 837 265 279 15 329 796 840 266 280 15 330 349 393 117 131 15 331 346 390 116 130 15 332 268 312 90 104 15 333 790 834 264 278 15 334 826 870 276 290 15 335 829 873 277 291 15 336 832 876 278 292 15 337 835 879 279 293 15 DRB5*0101 338 316 360 106 120 15 339 310 354 104 118 15 340 319 363 107 121 15 341 313 357 105 119 15 342 553 597 185 199 15 343 82 126 28 42 15 344 802 846 268 282 15 345 805 849 269 283 15 346 307 351 103 117 15 *Numbering according to SEQ ID NO: 1 **Numbering according to SEQ ID NO: 2

TABLE 4 NetMHCIIPAN predicted T cell epitopes in Api m 3 (only strong binders) Peptide Start No.* End No.* Start No.** End No.** Length Allele No. nucleic acid Nucleic acid amino acid amino acid amino acid DRB1*0101 1 523 567 175 189 15 2 625 669 209 223 15 3 511 555 171 185 15 4 316 360 106 120 15 5 889 933 297 311 15 6 652 696 218 232 15 7 1066 1110 356 370 15 8 751 795 251 265 15 9 961 1005 321 335 15 10 250 294 84 98 15 11 535 579 179 193 15 12 742 786 248 262 15 13 1018 1062 340 354 15 14 871 915 291 305 15 15 610 654 204 218 15 16 676 720 226 240 15 17 664 708 222 236 15 18 190 234 64 78 15 19 772 816 258 272 15 DRB1*0102 20 523 567 175 189 15 21 652 696 218 232 15 22 529 573 177 191 15 23 889 933 297 311 15 24 511 555 171 185 15 25 625 669 209 223 15 26 1075 1119 359 373 15 27 1066 1110 356 370 15 28 316 360 106 120 15 29 250 294 84 98 15 30 742 786 248 262 15 31 262 306 88 102 15 32 751 795 251 265 15 33 877 921 293 307 15 34 772 816 258 272 15 35 664 708 222 236 15 DRB1*0103 36 316 360 106 120 15 37 625 669 209 223 15 38 652 696 218 232 15 39 1066 1110 356 370 15 40 511 555 171 185 15 41 529 573 177 191 15 42 676 720 226 240 15 43 250 294 84 98 15 44 961 1005 321 335 15 DRB1*0104 45 523 567 175 189 15 46 529 573 177 191 15 47 889 933 297 311 15 48 652 696 218 232 15 49 625 669 209 223 15 50 250 294 84 98 15 51 511 555 171 185 15 52 1075 1119 359 373 15 53 751 795 251 265 15 54 1066 1110 356 370 15 55 742 786 248 262 15 56 262 306 88 102 15 57 316 360 106 120 15 DRB1*0105 58 523 567 175 189 15 59 625 669 209 223 15 60 511 555 171 185 15 61 316 360 106 120 15 62 889 933 297 311 15 63 652 696 218 232 15 64 1066 1110 356 370 15 65 751 795 251 265 15 66 961 1005 321 335 15 67 250 294 84 98 15 68 535 579 179 193 15 69 742 786 248 262 15 70 1018 1062 340 354 15 71 871 915 291 305 15 72 610 654 204 218 15 73 676 720 226 240 15 74 664 708 222 236 15 75 190 234 64 78 15 76 772 816 258 272 15 DRB1*0106 77 529 573 177 191 15 78 652 696 218 232 15 79 316 360 106 120 15 80 523 567 175 189 15 81 511 555 171 185 15 82 889 933 297 311 15 83 625 669 209 223 15 84 742 786 248 262 15 85 1066 1110 356 370 15 86 250 294 84 98 15 87 1075 1119 359 373 15 88 880 924 294 308 15 89 262 306 88 102 15 DRB1*0107 90 523 567 175 189 15 91 625 669 209 223 15 92 511 555 171 185 15 93 316 360 106 120 15 94 889 933 297 311 15 95 652 696 218 232 15 96 1066 1110 356 370 15 97 751 795 251 265 15 98 961 1005 321 335 15 99 250 294 84 98 15 100 535 579 179 193 15 101 742 786 248 262 15 102 1018 1062 340 354 15 103 871 915 291 305 15 104 610 654 204 218 15 105 676 720 226 240 15 106 664 708 222 236 15 107 190 234 64 78 15 108 772 816 258 272 15 DRB1*0108 109 523 567 175 189 15 110 625 669 209 223 15 111 511 555 171 185 15 112 316 360 106 120 15 113 889 933 297 311 15 114 652 696 218 232 15 115 1066 1110 356 370 15 116 751 795 251 265 15 117 961 1005 321 335 15 118 250 294 84 98 15 119 535 579 179 193 15 120 742 786 248 262 15 121 1018 1062 340 354 15 122 871 915 291 305 15 123 610 654 204 218 15 124 676 720 226 240 15 125 664 708 222 236 15 126 190 234 64 78 15 127 772 816 258 272 15 DRB1*0109 128 523 567 175 189 15 129 316 360 106 120 15 130 511 555 171 185 15 131 625 669 209 223 15 132 529 573 177 191 15 133 652 696 218 232 15 134 532 576 178 192 15 135 889 933 297 311 15 136 1066 1110 356 370 15 137 535 579 179 193 15 138 751 795 251 265 15 139 1018 1062 340 354 15 140 742 786 248 262 15 141 250 294 84 98 15 142 961 1005 321 335 15 143 610 654 204 218 15 144 676 720 226 240 15 145 871 915 291 305 15 146 664 708 222 236 15 DRB1*0110 147 523 567 175 189 15 148 625 669 209 223 15 149 511 555 171 185 15 150 316 360 106 120 15 151 889 933 297 311 15 152 619 663 207 221 15 153 652 696 218 232 15 154 751 795 251 265 15 155 1066 1110 356 370 15 156 1018 1062 340 354 15 157 535 579 179 193 15 158 961 1005 321 335 15 159 250 294 84 98 15 160 676 720 226 240 15 161 871 915 291 305 15 162 193 237 65 79 15 163 739 783 247 261 15 164 190 234 64 78 15 165 664 708 222 236 15 DRB1*0111 166 523 567 175 189 15 167 889 933 297 311 15 168 625 669 209 223 15 169 751 795 251 265 15 170 511 555 171 185 15 171 652 696 218 232 15 172 535 579 179 193 15 173 316 360 106 120 15 174 1018 1062 340 354 15 175 1066 1110 356 370 15 176 250 294 84 98 15 177 961 1005 321 335 15 178 610 654 204 218 15 179 190 234 64 78 15 180 676 720 226 240 15 181 871 915 291 305 15 182 193 237 65 79 15 183 91 135 31 45 15 184 1075 1119 359 373 15 DRB1*0112 185 523 567 175 189 15 186 625 669 209 223 15 187 511 555 171 185 15 188 316 360 106 120 15 189 889 933 297 311 15 190 652 696 218 232 15 191 1066 1110 356 370 15 192 751 795 251 265 15 193 961 1005 321 335 15 194 250 294 84 98 15 195 535 579 179 193 15 196 742 786 248 262 15 197 1018 1062 340 354 15 198 871 915 291 305 15 199 610 654 204 218 15 200 676 720 226 240 15 201 664 708 222 236 15 202 190 234 64 78 15 203 772 816 258 272 15 DRB1*0113 204 523 567 175 189 15 205 889 933 297 311 15 206 625 669 209 223 15 207 619 663 207 221 15 208 751 795 251 265 15 209 511 555 171 185 15 210 316 360 106 120 15 211 535 579 179 193 15 212 652 696 218 232 15 213 1066 1110 356 370 15 214 742 786 248 262 15 215 250 294 84 98 15 216 1075 1119 359 373 15 217 190 234 64 78 15 218 676 720 226 240 15 219 661 705 221 235 15 220 955 999 319 333 15 221 961 1005 321 335 15 222 1021 1065 341 355 15 223 871 915 291 305 15 224 208 252 70 84 15 225 847 891 283 297 15 226 91 135 31 45 15 DRB1*0114 227 523 567 175 189 15 228 625 669 209 223 15 229 316 360 106 120 15 230 511 555 171 185 15 231 652 696 218 232 15 232 751 795 251 265 15 233 889 933 297 311 15 234 1018 1062 340 354 15 235 535 579 179 193 15 236 1066 1110 356 370 15 237 961 1005 321 335 15 238 250 294 84 98 15 239 1075 1119 359 373 15 240 871 915 291 305 15 241 739 783 247 261 15 242 823 867 275 289 15 243 610 654 204 218 15 244 193 237 65 79 15 245 676 720 226 240 15 246 385 429 129 143 15 247 772 816 258 272 15 DRB1*0115 248 316 360 106 120 15 249 625 669 209 223 15 250 652 696 218 232 15 251 511 555 171 185 15 252 529 573 177 191 15 253 523 567 175 189 15 254 1066 1110 356 370 15 255 1018 1062 340 354 15 256 751 795 251 265 15 257 250 294 84 98 15 258 742 786 248 262 15 259 889 933 297 311 15 260 676 720 226 240 15 261 961 1005 321 335 15 262 610 654 204 218 15 DRB1*0116 263 625 669 209 223 15 264 529 573 177 191 15 265 523 567 175 189 15 266 1021 1065 341 355 15 267 751 795 251 265 15 268 889 933 297 311 15 DRB1*0117 269 523 567 175 189 15 270 625 669 209 223 15 271 511 555 171 185 15 272 316 360 106 120 15 273 652 696 218 232 15 274 889 933 297 311 15 275 751 795 251 265 15 276 1066 1110 356 370 15 277 1018 1062 340 354 15 278 250 294 84 98 15 279 535 579 179 193 15 280 961 1005 321 335 15 281 610 654 204 218 15 282 871 915 291 305 15 283 739 783 247 261 15 284 193 237 65 79 15 285 676 720 226 240 15 286 91 135 31 45 15 287 550 594 184 198 15 DRB1*0118 288 523 567 175 189 15 289 511 555 171 185 15 290 652 696 218 232 15 291 316 360 106 120 15 292 625 669 209 223 15 293 889 933 297 311 15 294 1066 1110 356 370 15 295 619 663 207 221 15 296 742 786 248 262 15 297 250 294 84 98 15 298 1075 1119 359 373 15 299 961 1005 321 335 15 300 751 795 251 265 15 301 535 579 179 193 15 302 871 915 291 305 15 303 664 708 222 236 15 304 1018 1062 340 354 15 305 676 720 226 240 15 DRB1*0119 306 523 567 175 189 15 307 625 669 209 223 15 308 511 555 171 185 15 309 316 360 106 120 15 310 889 933 297 311 15 311 652 696 218 232 15 312 1066 1110 356 370 15 313 751 795 251 265 15 314 961 1005 321 335 15 315 250 294 84 98 15 316 535 579 179 193 15 317 742 786 248 262 15 318 1018 1062 340 354 15 319 871 915 291 305 15 320 610 654 204 218 15 321 676 720 226 240 15 322 664 708 222 236 15 323 190 234 64 78 15 324 772 816 258 272 15 *Numbering according to SEQ ID NO: 1 **Numbering according to SEQ ID NO: 2

TABLE 5 Calculation of putative surface epitopes per protein size ratio Surface Surface Antigen Size Surface (Å²) epitopes* epitopes/kDa Amb t 5 4.3 kDa 2438.3 2.57 0.6 Api m 1 16-20 kDa 7606.4 8.0 0.4 Api m 2 43 kDa 15905.5 16.74 0.39 Api m 4 3 kDa 3885.7 4.09 1.36 Ara t 8 14.2 kDa 7080.1 7.45 0.52 Asp f 1 16.8 kDa 16037.6 16.88 1.0 Asp f 6 23.3 kDa 8793.2 9.26 0.4 Bet v 1 17.4 kDa 5215.3 5.49 0.32 Bet v 2 14.3 kDa 6493.9 6.84 0.48 Bos d 4 14.2 kDa 7246.9 7.63 0.54 Bos d 5 18.2 kDa 9546.5 10.05 0.55 Bos d 5 18.2 kDa 9618.4 10.12 0.56 Der f2 15.8 kDa 7785.2 8.19 0.52 Der p2 16 kDa 7588.8 7.99 0.5 Equ c 1 20 kDa 8907.4 9.38 0.47 Gal d 3 75.8 kDa 15952.9 16.79 0.22 Gal d 4 16.2 kDa 6951.3 7.32 0.45 Hev b 8 14 kDa 11982 12.61 0.9 Mus m 1 18.7 kDa 8943.5 9.41 0.5 Phl p 1 26.1 kDa 12145.6 12.78 0.49 Phl p 2 10.8 kDa 6099.5 6.42 0.59 Phl p 6 11.8 kDa 5429.5 5.72 0.48 Pru av 1 17.7 kDa 9742.8 10.26 0.58 Ves v 5 25.8 kDa 11657.1 12.27 0.47 Zea m14 11.7 kDa 5099.5 5.37 0.46 Average value 0.55 +/− 0.23 *Estimated IgE epitope area: 950 Å²

TABLE 6 Calculation of the average number of IgE epitopes on allergens Identified Possible IgE PDB Size Surface Size/ B-cell binding Antigen Protein Organism Common code (kDa) (Å²) Surface epitopes peptides Alt a 1 — Alt. alternata Fungi — 15.2 — — — 2 Ara h 1 Vicilin Arachis Peanut — 67.7 — — — 21 hypogaea Ara h 2 Conglutin Arachis Peanut — 17.5 — — — 10 hypogaea Asp f 1 Mitogillin Asp. Fungi 1AQZ 16.8 16037.6 1.0 16-17 13 fumigatus Asp f 2 — Asp. Fungi — 31.2 — — — 9 fumigatus Asp f 3 Peroximal Asp. Fungi — 18.4 — — — 7 protein fumigatus Asp f 13 Oryzin Asp. Fungi 28.7 — — — 5 fumigatus Bet v 1 PR10 Betulla Birch 1BV1 17.4 5215.3 3.3 5-6 verrucosa Bet v 2 Profilin Betulla Birch 1CQA 14.3 6493.9 2.2 6-7 3 verrucosa Bos d 5 b- Bos Cow 1B8E 18.2 9546.5 1.9  9-10 7 Lactoglobulin domesticus Bos d 5 b- Bos Cow 1QG5 18.2 9618.4 1.9  9-10 7 Lactoglobulin domesticus Cry j 2 Pectinase Cryp. Sugi — 42.2 — — — 4 japonica Gal d 1 Ovomucoid Gallus Chicken — 20.1 — — — 9 (8 IgG) domesticus Hev b 1 Elongaton Hevea Latex — 14.6 — — — 8 factor brasiliensis Hev b 3 SRPP Hevea Latex — 22.3 — — — 11 brasiliensis Hev b 5 — Hevea Latex — 15.9 — — — 11 brasiliensis Jun a 1 Pectate lyase Juniperus Cedar — 37.6 — — — 4 ashei Jun a 3 — Juniperus Cedar — 21 — — — 5 ashei Par j 1 Lipid transfer Parietaria Weed — 15 — — — 5 prot. 1 judaica Par j 2 Lipid transfer Parietaria Weed — 11.3 — — — 8 prot. 2 judaica Pen n18 Serine Pen. notatum Fungi — 52.4 — — — 9 protease 

1. A recombinant polypeptide capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera, wherein the polypeptide has a sequence identity of more than 90% to the amino acid sequence of SEQ ID NO:
 2. 2. The polypeptide of claim 1, wherein said polypeptide has the amino acid sequence of SEQ ID NO:
 2. 3. The polypeptide of claim 1, which is encoded by a naturally occurring nucleic acid of an insect from the order Hymenoptera.
 4. The polypeptide of claim 1, wherein the polypeptide contains one or more glycosylation sites having the sequence Asn-Xaa-Ser/Thr and, wherein one or more glycosylation site of the sequence Asn-Xaa-Ser/Thr has been mutated to a non-glycosylation site.
 5. A polypeptide capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera, and comprising at least 7 amino acids of a polypeptide having more than 90% sequence identity, or identical to a polypeptide selected from the group consisting of amino acid 26 to 99, 116 to 145, 185 to 223, 232 to 276 and 362 to 373 of the polypeptide shown in SEQ ID NO:
 2. 6. A polypeptide capable of binding to IgE from subjects allergic to venom of an insect from the order Hymenoptera, and comprising at least 7 amino acids of a polypeptide having more than 90% sequence identity, or identical to the polypeptide shown in SEQ ID NO: 2, except for the polypeptides from the group consisting of amino acids 1 to 34, 63 to 80, 100 to 115, 142 to 149, 168 to 176, 224-239 and 258 to 343 of the polypeptide shown in SEQ ID NO: 2 or except for the polypeptides shown in FIG.
 5. 7. A polypeptide selected from the group consisting of polypeptides consisting of amino acids 1 to 25, 146 to 152, 159 to 164, 168 to 184, 224 to 231, and 277 to 361 of the polypeptide shown in SEQ ID NO:
 2. 8. The polypeptide of claim 1, wherein the insect is a bee from the genus Apis.
 9. The polypeptide of claim 8, wherein the bee is Apis mellifera.
 10. The polypeptide of claim 1, wherein the polypeptide has acid phosphatase activity.
 11. The polypeptide of claim 1, wherein the polypeptide is linked to an additional polypeptide as a fusion protein.
 12. The polypeptide of claim 1, wherein the protein is non-glycosylated.
 13. The polypeptide of claim 11, wherein the additional polypeptide is selected from the group comprising a poly-Histidine tag, glutathione-S-transferase, β-galactosidase, a cytokine, an IgG-Fc or another Hymenoptera venom protein or antigenic fragment thereof.
 14. The polypeptide of claim 1, wherein the protein is expressed in bacterial or insect cells.
 15. The polypeptide of claim 14, wherein the protein is expressed in High5 or Sf9 cells.
 16. A method of producing a polypeptide of claim 1, comprising culturing a host cell comprising an expression vector comprising a nucleic acid encoding a protein for expression of said polypeptide and purifying said polypeptide.
 17. A method of treating a subject allergic to the venom of an insect from the order Hymenoptera, wherein the polypeptide of claim 1 is administered to said subject.
 18. (canceled)
 19. A method of diagnosing an allergy to the venom of an insect from the order Hymenoptera, comprising the steps of a) in vitro contacting a blood sample from a subject with a polypeptide of claim 1, and b) detecting binding of IgE antibodies to the polypeptide, wherein the detection of IgE antibodies bound to the polypeptide indicates said allergy.
 20. A pharmaceutical or diagnostical composition comprising a polypeptide of claim
 1. 21. The composition of claim 20, further comprising a suitable adjuvant or expedient or further polypeptides from the venom of an insect from the order Hymenoptera.
 22. A method of diagnosing an allergy to venom of an insect from the order Hymenoptera, comprising the steps of a) performing the method of claim 16, b) contacting the polypeptide obtained by the method of step a) in vitro with a blood sample from a subject, and c) detecting binding of IgE antibodies to the polypeptide, wherein the detection of IgE antibodies bound to the polypeptide indicates said allergy.
 23. A method of claim 22, wherein the method differentiates between allergies to venom of different insects of the order Hymenoptera.
 24. A method of preparing a composition for diagnosing an allergy to venom of an insect from the order Hymenoptera comprising the step of performing the method of claim
 16. 25. A method of preparing a composition for treating subjects allergic to the venom of an insect from the order Hymenoptera, comprising the step of performing the method of claim
 16. 